Search

A subpopulation of cancer cells, called cancer stem cells (CSCs), are thought to be responsible for cancer relapse and metastasis, phenomena which are the cause of the majority of cancer deaths. CSCs are highly treatment resistant and are not removed by current treatments at their administered doses. New chemotherapeutics are needed which can remove CSCs alongside bulk tumour cells at safe doses. A promising strategy to eradicate entire tumours, including CSCs, is immunogenic cell death (ICD). ICD is a mode of cell death which stimulates the immune system to further remove cancerous cells. A CSC-active ICD agent may be able to directly eradicate bulk breast cancer cells and CSCs and stimulate the immune system to remove residual cells. ICD occurs following oxidative stress of the endoplasmic reticulum (ER). Redox active copper(II) complexes have been shown to elevate intracellular ROS levels. If directed to the ER, copper(II) complexes may be capable of oxidatively stressing the ER, triggering ICD. This project aims to develop novel copper(II) complexes which can target the ER, elevate intracellular ROS levels and induce ICD in breast CSCs. This thesis describes the synthesis, characterisation, and evaluation of the biophysical and anti-CSC properties of the complexes. Novel copper(II) complexes with improved stability and potency compared to previous anti-breast CSC copper(II) complexes were developed. Complexes 4 and 17, containing 4,7-diphenyl-1,10-phenanthroline, were found to induce ICD in breast CSCs following oxidate ER stress. Complex 15, containing 3,4,7,8-tetramethyl-1,10-phenanthroline, was found to induce autophagic cell death in breast CSCs. Complex 13, containing 1,10-phenanthroline, was found to induce apoptosis in breast CSCs. The phenanthroline ligand present in these complexes determines the mode of breast CSC death. Complexes 19-23 and 26, containing sulphonamide-functionalised terpyridine ligands, were found to increase intracellular ROS levels, partially induce ER stress, and kill breast CSCs via caspase independent apoptosis.

Neural stem cells (NSCs) in the brain can be reactivated to exit quiescence and generate new cells in response to injury or disease. Determining the specific signals involved in NSC reactivation is essential to understanding neural regeneration. Investigating NSCs in *Drosophila melanogaster* provides a powerful model due to ease of identification of quiescent NSCs and a plethora of genetic and molecular tools available to study the nervous system *in vivo*. During development, *Drosophila* NSCs reactivate in response to dietary amino acids sensed by the fat body, a sensor organ analogous to liver and adipose tissue in mammals. Previously unknown signals from the fat body are transmitted to the bloodbrain barrier (BBB) glia on the surface of the brain. The glia secrete insulin-like peptides, which are received by the insulin receptor on NSCs, inducing their reactivation. To identify the fat body-derived signals sent to the brain, I used Targeted DamID to generate transcriptional profiles of the fat body under fed and starved conditions. I discovered upregulation of *dpp (decapentaplegic)*, a transforming growth factor (TGFβ) secreted morphogen, after feeding. Knockdown of Dpp in the fat body severely impaired NSC reactivation. Similarly, knocking down a key receptor for Dpp, Tkv (Thickveins), in the BBB glia led to severely impaired NSC reactivation. To compare gene expression in glial subtypes under fed and starved conditions, I generated single-cell RNA sequencing datasets. I found Dpp to be upregulated in BBB glia in response to feeding and functional experiments showed that Dpp signalling in the glial niche is required for NSC reactivation. The presence of Medea transcription factor binding sites at the dIlp6 locus and impaired NSC reactivation when Medea is knocked down in the BBB glia suggest that Dpp signalling may regulate expression of dIlp6. Knockdown of Tkv in the NSCs themselves did not affect reactivation, suggesting autocrine Dpp signalling in the BBB glia in response to interorgan Dpp signalling from the fat body to the brain. The findings may help understand how to induce neural stem cell proliferation after brain damage or neurodegeneration.

Background: Microtubule associated proteins are a group of proteins well characterised for their role in the stabilisation and polymerisation of microtubules, but they have been increasingly demonstrated to have a more diverse role within cells. Tau is a microtubule associated protein, well studied for its pathological role in Alzheimer's disease. Recent studies have provided evidence that tau is also expressed in the epidermal compartment of the skin. However, the precise roles of tau during epidermal stratification and differentiation are currently unknown. Aims: This study aims to investigate the role of tau in epidermal stratification and differentiation and its function in cutaneous squamous cell carcinoma (cSCC). Methods: Immunostaining was used to investigate tau expression in clinical samples, 2D cultures and 3D skin equivalents. To explore the functional significance of tau during keratinocyte differentiation and stratification, siRNA and shRNA were used to downregulate tau expression in keratinocytes and the cSCC cell line, A431. Gene and protein expression were assessed using RT q-PCR and Western blotting, and changes in cytoskeletal organisation were determined using immunofluorescence. The overexpression of tau was conducted through the generation of stable cell lines with a doxycycline inducible overexpression of tau. The functional significance of tau overexpression was determined using a combination of RT q-PCR, Western blot and immunofluorescence analyses of 2D epidermal keratinocyte culture, MatTek© 3D epidermal models and cSCC cells in vitro. Co-Immunoprecipitation (CoIP) and subsequent proteomic analysis were performed to identify tau binding partners in growing and differentiated keratinocytes. Results: In healthy human skin, tau expression was upregulated in the differentiated population of suprabasal keratinocytes and displayed a distinct isoform specific expression throughout the epidermis. Overexpression of tau in vitro was found to initiate keratinocyte differentiation in 2D culture and 3D epidermal models. CoIP and subsequent proteomic analysis identified a discrete pattern of tau interactions in keratinocytes cultured under growing and differentiated conditions, with a specific subset of nuclear, cytoplasmic and membrane associated proteins identified to interact with tau through different stages of keratinocyte differentiation. Finally, in cSCC tau expression was linked to the differentiation status of the tumours. Furthermore, the downregulation and overexpression of tau was sufficient to induce changes to cytoskeletal organisation and cellular differentiation in cSCC in vitro. Conclusions: This study revealed that tau can orchestrate epidermal cell fate, differentiation and stratification in healthy human skin and cSCC. These findings are therefore anticipated to be a starting point for more functional research into the binding partners of tau during epidermal stratification, differentiation and their subsequent function in cancer.

Introduction: The development of small-diameter vascular grafts remains a key challenge in Tissue Engineering. Over the last two decades, strides have been made on the biofabrication of vessel mimics with similar biophysical characteristics to those of mammalian vessels but with only modest success. These vessels lack remodelling and regenerative capacity. This thesis aims to use induced pluripotent stem cells (iPSCs) and functionalised biomaterials to produce patientspecific bioengineered vessels. Materials and Methods: Human iPSCs were first differentiated into mesenchymal stem cell (MSC), designated as iMSCs, with supplement of SB431542, a TGF-ß1 pathway inhibitor. The iMSCs were further differentiated into vascular smooth muscle cells (VSMCs) via platelet-derived growth factor (PDGF)-BB and transforming growth factor beta 1 (TGF-ß1) supplementation. Human mesenchymal stem cells derived from bone marrow (hMSCs-BM) from a commercial source were used for optimisation of differentiation protocols and quality control of MSCs. Vascular scaffold was produced using electrospinning of poly-L-lactide (PLLA) and coated with silk fibroin. HMSCs-BM and iMSCs were grown on the scaffold to evaluate the biocompatibility of the materials. HMSCs-BM and iMSCs were seeded in silk fibroin coated electrospun PLLA sheet for in-scaffold differentiation to obtain VSMCs. The cell-laden scaffold sheets were wrapped around a 3 mm diameter stainless steel rod and fixed with an alginate sealing to form a tubular shaped vascular graft. Results: During the 34-36 days differentiation, the human iPSCs from three different cell lines gradually gained a fibroblast-like morphology, had increased expressions of MSC marker genes CD73, CD90 and CD105 and stained positive for these markers via immunofluorescence. Flow cytometry of the iPSC derived iMSCs also showed an increase in cell populations positive for these markers during differentiation. Thereafter, hBM-MSCs and iMSCs were successfully differentiating into VSMCs during a 9-day culture in PGDF-BB and TGF-ß1 supplemented medium. The MSC-VSMCs express VSMC marker genes of a-SMA and CNN1, SM22 and MYH-11, which were confirmed by immunofluorescence staining. The PLLA silk fibroin coated porous scaffolds was compatible for MSCs adhesion and was best at supporting cell growth during a 10-day culture period over the non-silk fibroin coated pristine and porous scaffolds. The engineered tubular constructs derived from cell-laden PLLA silk fibroin coated scaffolds showed to have cell populations positive for VSMC markers a-SMA and CNN1. Furthermore, these constructs displayed mechanical properties comparable to those of autologous vascular grafts used for coronary artery bypass graft (CABG) like saphenous vein and internal mammary artery. Discussion/Conclusion: An optimised protocol for iPSC to VSMC differentiation via MSCs was established. Silk fibroin coating on electrospun porous PLLA scaffold demonstrated an enhanced biocompatibility to MSCs. In-scaffold differentiation of VSMCs from iMSCs was successful which further produced a cell-laden small diameter vascular graft. The fabricated blood vessel mimics have native like biological and mechanical features. Future work will concentrate on the conjugating growth factors to the silk fibroin coating and evaluating the optimal release from these scaffolds to promote VSMC differentiation in situ from iMSCs.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the progressive loss of upper and lower motor neurons, eventually culminating in patient paralysis and death via asphyxiation. At the time of diagnosis, approximately 50% of patient motor neurons are predicted to have degenerated, resulting in a prognosis of 3-5 years. While most incidences of ALS are sporadic (~90%) with no obvious genetic constituent, approximately 10% of cases are inherited in a dominant manner and are referred to as familial ALS. At the present time there is no cure for ALS and current pharmacological treatments, such as Riluzole, operate with limited efficacy. As such, gaining further insight into pathomechanisms driving ALS is paramount for the development of novel and effective therapeutics. The C9ORF72 hexanucleotide repeat expansion is attributed to 40-50% of familial and approximately 10% of sporadic ALS cases. Investigating pathomechanisms driving familial cases of C9-ALS could elucidate neurodegenerative mechanisms central to both familial and sporadic cases. The aim of this thesis was to investigate transcriptional perturbations in C9-ALS to elucidate de novo candidates rendering motor neurons particularly susceptible to pathology. Evidence from previous studies suggested that nociceptors of ALS patients remain functionally viable due to the presence of non-neuropathic pain in approximately 80% of patients. We subsequently developed a novel protocol to efficiently generate functional sensory neuron cultures predominantly consisting of spinal nociceptors (~75%). Utilizing our protocol, we compared the transcriptomes of C9-ALS nociceptors with C9-ALS motor neurons which elucidated dysregulation of synaptic genes in motor neurons, potentially suggesting increased susceptibility of motor neurons to excitotoxic mechanisms. Finally, we utilised CRISPR systems employing dCas9 fused to epigenetic effectors to selectively demethylate the C9ORF72 repeat expansion and proximally associated regions to investigate epigenetic contributions to pathology. We observed an increase in transcript variant 3 expression and retention of intron 1 was observed upon demethylation of the repeat expansion. While increased expression of C9ORF72 could exacerbate pathomechanisms driven by RNA foci and accumulation of DPR proteins, deficits in synaptic function could be attenuated by reducing C9ORF72 haploinsufficiency. In summary, the work presented in this thesis outlines novel approaches to investigating the mechanisms driving pathology in C9-ALS with the hope of elucidating de novo candidates for therapeutic targeting. Furthermore, the ability to generate functional nociceptors broadens the scope of pain research permitting in vitro experiments to understand the mechanics of pain in human cells.

Embryogenesis entails complex signalling interactions between embryonic and extraembryonic lineages. Improved embryo culture techniques have recently allowed human embryos to be cultured in vitro past the point at which they would implant in vivo, until the internationally accepted culture limit of 14 days. Nonetheless, the reliance on surplus in vitro fertilised (IVF) embryos, along with the ethical and legal considerations associated with genetic manipulation of human embryos, present barriers to the study of postimplantation human development. However, human stem cell-based embryo models offer a system that is amenable to genetic and molecular manipulation. Within mouse development, the anterior visceral endoderm (AVE), formed from the extraembryonic endoderm, plays a crucial role in restricting primitive streak formation to the posterior epiblast, thereby establishing the anterior-posterior axis of the mouse embryo. Recent studies of human embryos cultured to postimplantation stages have alluded to the presence of a human equivalent of the mouse AVE in the anterior hypoblast, the hypoblast being the portion of the human yolk sac that lies adjacent to the epiblast. However, the functional role of such a structure within the context of human development is yet to be understood. In vitro stem cell modelling of the human yolk sac- epiblast interaction offers a system through which the role of the yolk sac in postimplantation epiblast morphogenesis can be interrogated. Consequently, the aim of this study was to derive yolk sac-like cells (YSLCs) that could be used to model human epiblast-yolk sac crosstalk in vitro. Following a screen of developmentally relevant signalling pathway activators, this project identified that the coactivation of NODAL, WNT and JAK/STAT signalling via ACTIVIN-A, CHIRON, and leukaemia inhibitory factor (LIF) (ACL) induced an endodermal fate in human pluripotent stem cells (hPSCs). Functional and transcriptional analysis of ACL-treated cell populations revealed that the pluripotent state harboured by hPSCs determines the type of endoderm that ACL treatment induces. Human embryonic stem cells (hESCs), which harbour a primed pluripotent state, were found to respond to ACL by adopting a definitive endoderm-like fate, whereas Rset hPSCs, which harbour a pluripotent state that is an intermediary between naïve and primed, gave rise to a yolk sac-like population. Accordingly, Rset hPSCs treated with ACL were coined YSLCs. YSLCs were found to express inhibitors of BMP, WNT and NODAL signalling, while YSLC conditioned medium was shown to inhibit downstream activity of BMP and WNT signalling in hESCs in both 2D and 3D culture conditions. Similarly, YSLC conditioned medium supported the coexpression of pluripotency and anterior ectoderm markers in hESCs, at the expense of posterior epiblast markers. These results indicated that YSLCs share functional characteristics with the mouse AVE, and also potentially the human anterior hypoblast. Collectively, therefore, this project proposes YSLCs as a tool for studying human epiblast-yolk sac crosstalk.

Amyotrophic lateral sclerosis (ALS) is an incurable, rapidly progressive and fatal neurodegenerative disorder, characterised by loss of upper and lower motor neurons (MNs). Approximately 10-20% of ALS cases are familial, of which the C9orf72 (G4C2)n > 30 hexanucleotide repeat expansion mutation is the commonest known cause in the western world. The finding that familial ALS is clinically and pathologically indistinguishable from sporadic ALS supports the study of monogenetic causes to better understand common pathogenic mechanisms. Thus, human induced pluripotent stem cell (iPSC) derived MN experimental platforms, combined with paired gene-edited isogenic control lines, provide a powerful approach, both to identify early-stage disease-driving mechanisms, and establish causality between a given mutation and phenotypes. Such in vitro disease modelling is complemented by in vivo approaches, particularly those that harness technologies interrogating molecular neuropathological signatures in human post-mortem tissue. The primary aim of this PhD was to study the MN cell-autonomous pathogenic mechanisms of the C9orf72 repeat expansion against an isogenic control background using a human model, adopting cellular, molecular and bioinformatics techniques. I establish that C9orf72 MNs have dysfunctional axonal homeostasis, with aberrations both in their axonal morphology (reduced neurite length) and function (impaired fast axonal transport of mitochondrial cargo). Axonal dysfunction is a common phenotype in neurodegenerative disorders, including in ALS, where MNs have axons extending up to a metre long. The maintenance of axonal function is a highly energy-demanding process, raising the question of whether MN cellular energetics is perturbed in ALS, and whether its recovery promotes axonal rescue. I show that these axonal phenotypes are associated with concomitant metabolic dysfunction, owing to defective mitochondrial respiration. Unbiased RNA-sequencing revealed reduced expression of electron transport chain transcripts, encoded by mitochondrial DNA, the copy number of which was unaltered. Critically, I show, through neuropathological analysis of patient post-mortem tissue, that this transcriptomic dysregulation is selective to anterior horn spinal (motor) neurons, and is absent in dorsal horn spinal (sensory) neurons, with corresponding alterations reflected at the level of protein expression. Through manipulation of this molecular mitochondrial loss-of-function signature, leading to therapeutic rescue of the observed dysfunctional axonal homeostasis, I determine a novel causal relationship between axon dysfunction and contributory metabolic dysfunction in C9orf72-ALS. Collectively, my experimental data show that loss of mitochondrial function is a key mediator of axonal dysfunction in C9orf72-ALS, and that boosting MN bioenergetics is sufficient to restore axonal homeostasis, opening new potential therapeutic strategies for ALS that target mitochondrial function. These data build on preclinical data from animal models in ALS (albeit largely biased by a preponderance of data from SOD1 models), which I systematically reviewed and meta-analysed, showing that targeting mitochondrial dysfunction may prolong survival, particularly if the intervention is administered early.

Viscoelasticity encompasses the characteristics of both the shape-conserving elasticity and the shock-absorbing viscosity. The extracellular matrices (ECM), or the protein-polymer scaffolds surrounding the cells in our body, possess unique tissue-dependent viscoelasticity to protect and provide mechanical queues to the nearby cells. Viscoelasticity in polymeric hydrogels can be achieved in several ways, one of which is by incorporating the host-guest supramolecular components. The following works explore the application of cucurbit[8]uril (CB[8])-based host-guest chemistry to achieve tuneable viscoelasticity in hydrogels. First, an ECM-mimetic CB[8]-based biomaterial tuned to the viscoelasticity of the lung is developed for stem cell engraftment into the solid organs. The crosslinker designed with a MMP-cleavable sequence enabled the enzymatic and cell-mediated changes in the gel mechanical properties, supported the organoid culture, and facilitated the functional engraftment and differentiation of the lung stem cells in the mouse lungs, highlighting the importance of tuning the viscoelasticity and cell-responsiveness of stem cell-carrying biomaterials for engraftment into solid organs. The second work reports of biofabricating the flexible electronics through a parylene surfacemodification chemistry and a novel hybrid network consisting of CB[8] and 1-benzyl-3vinylimidazolium (BVI) host-guest crosslinkers and gelatin-methacrylate (GelMA). The gel was designed to match the stress relaxation profile of the brain, facilitating a more brain ECM-mimetic biomaterial that is resistant to the high strain of the flexible neural devices. Combined with the ability to photo-pattern and combine with the different types of cells, this work presented the potential ways of multiplexing different types of biomaterials and cells to augment the bioelectronics functionalities, opening a new era of regenerative bioelectronics. The final work describes a new coumarin-based monomer that can be incorporated into the various free-radical-polymerised and-crosslinked materials to achieve reversibly phototuneable viscoelasticity. Harnessing coumarin's UV wavelength-dependent cyclo-additionvi and-reversion, an extraordinary range ( 0.01 seconds to 1 million seconds) of stress relaxation half time (τ1/2) and storage modulus (100-10,000 Pa) were achieved with UV irradiation alone without changing the molecular composition. Unlike the previous approaches of directly functionalising the coumarin onto the polymer backbones, generating a CB[8]-coumarin monomer provided a powerful platform to augment the existing materials to possess reversibly tuneable viscoelasticity.

Neural stem cells produce all the cells of the nervous system and regulation of proliferative behaviour is crucial for proper brain development. Over-proliferation of NSCs can also lead to disease, resulting in the formation of tumours. There is a delicate balance between NSC proliferation, differentiation and quiescence. Studying the mechanisms that regulate this balance is important for understanding development and disease, providing a basis for new therapeutic strategies for brain injury or cancer. In Drosophila melanogaster, a small pool of NSCs produce all of the 200 000 neurons present in the adult brain. The precise mapping of NSCs in the brain, short developmental time, genetic tractability and wide variety of experimental tools make Drosophila an ideal model for investigating NSC behaviour. My project utilises single-cell transcriptomics to study two aspects of brain development: 1) the regulation of developmental NSC quiescence and its relevance to glioblastoma, and 2) identifying novel players regulating the temporal identity of neural progenitors and how this generates neuronal diversity. NSCs in the larval Drosophila brain undergo quiescence, a reversible, cell-cycle arrested state, after a period of neurogenesis and reactivate in response to extrinsic signals, namely feeding. In order to investigate how quiescence is regulated, I used scRNA-seq to profile quiescent NSCs. One of the genes found to be enriched during quiescence was tis11, involved in regulating RNA stability. Functional studies of tis11 suggest that it is involved in the maintenance of quiescence. Interestingly, ZFP36, the orthologue of tis11 is also expressed in human glioblastoma cells that are slow-cycling and express genes characteristic of mammalian NSC quiescence. Glioblastoma is a cancer with poor prognosis, as chances of tumour recurrence are high. This may be due to the presence of a subpopulation of cancer stem cells which divide slowly and are therefore resistant to conventional chemotherapy. Expression of ZFP36 (involved in developmental quiescence) in glioblastoma cells suggests that they are quiescent and may be a source of tumour recurrence. Understanding the mechanisms of tis11 during developmental quiescence may therefore lead to a better understanding of action in the context of glioblastoma and inform better treatment strategies. Brain development requires different subtypes of neurons to be produced at the right time and in the right numbers, in order to achieve functional connectivity. The Drosophila brain has a class of NSCs that produce intermediate neural progenitors (INPs) that transition through a 'temporal cascade'. The temporal cascade is a series of sequentially expressed transcription factors that cross-regulate one another and determine the subtypes of neuronal progeny that are produced. The INP temporal cascade was originally identified by antibody screens but the cross-regulatory interactions were not comprehensive, suggesting that there are unknown players yet to be identified. Using a newly developed technique from our lab, NanoDam, the genome-wide binding targets of the previously identified temporal factors were profiled in the INPs, in their endogenous windows. NanoDam enables profiling of chromatin-binding proteins with just one genetic cross. Combined with scRNA-seq of the INPs, two new candidate temporal factors were identified: Homeobrain and Scarecrow. These novel temporal factors, in addition, have roles in regulating NSC longevity. These NSCs divide in a similar manner to the outer radial glia of the mammalian brain and both factors have orthologues, namely ARX and NKX2.1. Remarkably, we showed that these factors have conserved roles in the NSCs of the visual system, which have a different developmental origin.

Neurodegeneration is a pathology shared by a varied class of diseases, and many of the mutations that are known to cause such diseases have been linked to protein aggregation and autophagy dysfunction. Improvements to gene editing and neuronal differentiation strategies have enabled the derivation of in vitro disease models using human iPSC-derived neurons to provide a more accurate understanding of how disease mutations affect neuronal health. I first sought to model the polyglutamine disease spinal and bulbar muscular atrophy (SBMA), detailed in Chapter 4. Using iPSCs derived from a healthy donor and an SBMA patient, the CAG repeat of the endogenous androgen receptor (AR) gene was CRISPR-edited to encode a series of lengths or an early stop codon. However, AR expression was silenced upon transcription factor-mediated differentiation to a lower motor neuron-like state, and chemical differentiation prevented ligand-induced AR nuclear translocation. Deriving the cell lines in this work highlighted that purification of transgenic cells is a key bottleneck to gene editing. I therefore adapted a synthetic marker gene that presents a streptavidin binding peptide (SBP) tag on the extracellular membrane, detailed in Chapter 3. Expression of this tag in iPSCs enabled transient fluorophore staining and effective sorting of mixed populations with magnetic streptavidin beads. Finally, Chapter 5 establishes L1CAM as a novel autophagy modulator; iPSC-derived neurons showed that reduction of the L1CAM transcript with shRNAs, but not genetic knockout of the L1CAM protein, is sufficient to reduce transcription of the ATG8 gene family, which are core components of macroautophagy. This work exemplified both the strengths and weaknesses of iPSC-derived neurons; namely, they are tractable and able to recapitulate neural phenotypes, but deriving new model lines requires a high initial investment, so adequate proof-of-concept is crucial.

The liver is the primary organ involved in drug detoxification, hence being highly exposed to the risk of drug induced liver injury (DILI). DILI is the main cause for acute liver failure in western countries and post-marketing withdrawal of drugs. This is highly due to the poor identification of potential human hepatotoxins during the preclinical phase of drug discovery. It is not surprising that interspecies difference between animal models could generate diverse biological responses compared to the human liver. Similarly, the current-state-of-the-art human in vitro hepatic systems lack the cellular spatial arrangement and hepatocyte functionality observed in vivo. Identification of an in vitro hepatic system capable to reproduce the essential features of the human liver in vivo is thus beneficial to better understand DILI and how it is triggered. Historically, maintenance of primary hepatocyte in vitro has been challenging due the intrinsic finite proliferative capacity of these cells, the shortage of donor livers, and the inability to maintain their in vivo structural and functional identity in vitro. All of the above hurdles have been addressed with the advent of 3D epithelial structures called organoids, yet its use in the liver safety field is currently unexplored. Liver organoids have the great advantage to be highly proliferative, to maintain the identity of the tissue of origin, and to retain the bipotential ability to differentiate into hepatocyte organoids. In this Thesis, we generated an enhanced hepatocyte organoid system through the directed stepwise differentiation in vitro of progenitor liver organoids using chemically defined culture conditions. Validation of these organoids in the context of DILI, showed structural and functional formation of tubular like structures called bile canaliculi which are essential morphological structures for the hepatocytes to secrete drugs/metabolites and bile from the intracellular compartment. Drug distribution mapping analysis revealed the ability of these organoids to uptake exogenous compounds. Therefore, we examined the potentiality of the hepatocyte organoids to detect known human hepatotoxins, which resulted in the obstruction of hepatic bile clearance and downregulation of transmembrane proteins in a process called cholestasis. Furthermore, formation of typical features of drug-induced steatosis were observed upon treatment with steatotic drugs. In summary, our findings highlight how this novel differentiated organoid system could be a relevant human in vitro system with the potential to be incorporated into an initial drug safety screening, either as a replacement or complementary hepatic system.

The first cell fate decision in development occurs at the blastocyst stage with the emergence of the trophectoderm (TE) and the inner cell mass (ICM). The TE is the precursor population of all major placental cell types. Reflecting this developmental plasticity, trophoblast stem cells (TSCs) can be derived from the TE of mouse blastocysts. TSCs have proven an invaluable research tool to study processes of early placentation in vitro. Despite the placenta's central role in reproduction, our understanding of the regulatory networks that orchestrate TSC self-renewal and differentiation remains incomplete. In this project, I characterised an epigenetic modifier, KDM1B, for its role in TSC self-renewal and differentiation. I identified this factor as a putative novel regulator of trophoblast stem cell fate and in vitro differentiation from transcriptomics data as its expression is markedly induced at the onset of differentiation. Furthermore, Kdm1b had been implicated in mouse development and placentation, via directing DNA methylation of maternal imprints in the oocyte. KDM1B is a histone lysine demethylase whose activity is directed to H3K4me1 and H3K4me2, particularly within the gene body of actively transcribed genes. By generating CRISPR-Cas9-mediated knockout TSCs ablated for Kdm1b, I show that Esrrb is consistently down-regulated but Gcm1 is up-regulated in Kdm1b-/- TSC clones as measured by RT-qPCR, indicative of precocious differentiation into the syncytiotrophoblast lineage. By performing a large cohort of integrated genome-wide analyses, notably RNA-seq and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) for the relevant histone modifications H3K4me1, H3K4me2, H3K4me3 and H3K36me3, I show that KDM1B regulates intragenic H3K4me1-marked enhancers, while not impacting H3K4me3. Remarkably, KDM1B null trophoblast cells also show an increased instability of chromosome 13, the same chromosome on which Kdm1b itself is located. This chromosome hosts several gene families that arose from gene duplication events, with vital roles in trophoblast development. The instability of chromosome 13 that gave rise to these gene families is apparently exacerbated by loss of KDM1B, or by CRISPR Cas9-induced cutting of the chromosome. In addition to interrogating the role of KDM1B during differentiation, I utilised the transcriptomic and ChIP-seq data to identify several trophoblast-specific transcripts via location of distal H3K4me3 peaks. Using previously published ChIP-seq data in combination with my own, I link enhancer activity in stem cells to gene expression throughout differentiation. Finally, I identify a striking and novel redistribution of H3K36me3 in 5 day differentiated trophoblast cells, to the promoter regions of expressed genes. In conclusion, this work presents an in-depth analysis of the transcriptional and epigenomic rearrangements that occur both in WT trophoblast and as a consequence of Kdm1b deletion. These data lend important insights into the functions of this epigenetic modifier in the fine-tuning of the transcriptional networks that direct TSC self-renewal and differentiation.

Organoid systems have revolutionised the study of adult stem cells and organ homeostasis. However, there have been limited attempts to use the technology to study human organ development. The work of this thesis established two novel hepatic organoid systems; one based upon the archetypal liver stem cell, the hepatoblast, whilst the other was based upon a hitherto undescribed biliary progenitor cell. The work continued with exploration of the development of the human intestine, characterising how intestinal organoids develop in vitro, and going on to assess human intestinal maturation in vivo at single cell resolution. First, the developing human liver was characterised during the first trimester and the cellular composition at this crucial developmental timepoint was investigated at single cell resolution. The hepatoblast organoids (HBO) were then demonstrated to retain their hepatoblast profile in culture and demonstrated the functional capacity of this fetal cell-type. Exploiting this new model, the molecular processes of differentiation were then explored as the HBO were demonstrated to have bipotential capacity to generate hepatocytes and cholangiocytes. Furthermore, these organoids were then transplanted into mice and were able to differentiate into complex functional tissues. Next, a newly discovered stem cell was characterised and termed the 'biliary progenitor cell'. This new cell type was able to generate three-dimensional organoids, fetal biliary organoids (FBO), that could be transplanted into mice and provide insight into biliary development. Next induced pluripotent stem cell (iPSC)-derived and primary fetal intestinal derived organoids were characterised against purified primary intestinal epithelial tissue, demonstrating a dynamic transcriptome of fetal organoids in vitro and a lack of tissue specificity of iPSC-derived organoids. The fetal and paediatric intestine were assessed at single cell level to assess the cell types and developmental pathways involved in the process of development, and this   information was then utilised to try to model development in vitro using small molecule stimulation of fetal intestinal organoids. The work described in this thesis develops our understanding of human endodermal organ development. The newly described organoid models present an exciting opportunity to model human liver development, generate cells for drug-screening, and offer the potential for cell-based therapies, whilst the exploration of the human intestine during development may aide our understanding of how such pathways may be abrogated in developmental defects, or reverted to during disease states.

Fasciolosis, a debilitating disease caused by flatworm parasites belonging to the genus Fasciola, is a prominent threat to global food security due to its widespread infection of agriculturally significant ruminants. Recent studies have identified a population of proliferative cells within Fasciola hepatica (Fasciola proliferative cellsFPCs) that resemble neoblast-stem cells, both morphologically and behaviourally. These cells are the only proliferating cells in Fasciola hepatica and can be localised using thymidine analogue 5-Ethynyl-2 ́-deoxyuridine (EdU). This thesis further characterises these neoblast-like stem cells, investigating their dynamics, supportive roles in growth and development and identifying putative regulatory effectors. This study reports increased juvenile growth, which correlated with increased proliferation of these stem-like cells, in juveniles maintained in chicken serum supplemented media (CS+). The comparison of FPCs between in vitro maintained and ex vivo maintained juveniles further supports the link between FPCs and juvenile development, with the larger, more developed ex vivo juveniles exhibiting significantly more proliferating cells. FPCs were found to be radiation sensitive enabling us to selectively deplete them and examine downstream effects. Using an RNA interference (RNAi) platform, the function of a key effector (fgfrA) was investigated and silenced juveniles exhibited a significant reduction in the number of EdU+ cells. The findings of this study highlight the stem-like characteristics of FPCs, their facilitative roles within fluke growth and indicate their importance to fundamental fluke biology. The disruption and dysregulation of this cell population may hold the key to developing novel control therapies.

Traditionally, cell studies have focused mainly on the effects of biochemical signals on cellular behaviour; however, there is a growing interest in investigating the effects of physical cues on the behaviour of the cells. Development of fabrication techniques has allowed researchers to design topographical cues at the micro and nanometer scale. In this study, we fabricated fibronectin stripes, which mimic some of the frequent patterns that cells are exposed to in their natural microenvironment, and studied the effects of these patterns on 3T3 fibroblasts and human mesenchymal stem cells which are of significant importance in the field of regenerative medicine and tissue engineering. Our results show that cells align and elongate in the direction of the patterns and the morphological features of cell, nucleus and architecture of force-bearing stress fibers of the actin cytoskeleton are all highly dependent on the stripe width. 3D confocal measurements showed that the cell thickness and nuclear shape is regulated through the different arrangement of perinuclear actin stress fibers depending on the stripe sizes. We also observed that when the size of stripes (5 μm) is significantly smaller than the size of cell nuclei (subnuclear stripes), cells exhibit 3 different morphological responses to the patterns. Minimal confinement, branching on the stripes and elongation on a single stripe. The majority of cells and nuclei align themselves with the subnuclear stripes; however, a substantial number of hMSCs nuclei are elongated perpendicular to the direction of the pattern, a finding which we explained using a geometrical model based on cell size. The behaviour of 3T3 cell lines and the hMSCs were compared for the different types of pattern. We designed setups that allow us to perform Raman microspectroscopy both in 2D and 3D topographical patterns in order to be able to study their morpho-chemical effects on cells. We also genetically modified 3T3 fibroblasts to express fluorescent-tagged proteins that visualize the actin cytoskeleton and the nucleus which allow us to perform live cell imaging to study the dynamics of cell behaviour on topographical patterns.

The early stages of development are characterised by profound transcriptional and epigenetic reprogramming. The most striking example is the global loss of DNA methylation patterns on the maternal and paternal genomes that is precipitated upon formation of the zygote. This is coincident with the establishment of the inner-cell mass, a pluripotent population of cells that give rise to the embryo. The necessity to reset the epigenetic landscape to attain this state however requires further investigation, as are the mechanisms that lead to these changes. Using a mouse embryonic stem culture media, referred to as 2i, that models this developmental period, I set out to uncover mechanisms that may contribute to the remodelling of DNA methylation. In contrast to traditional serum-based mouse embryonic stem cell culture media, 2i maintains DNA methylation at a significantly lower level. The use of 2i and serum cultures therefore provided a means to investigate the regulation of the different DNA methylation landscapes. The overarching focus has been to understand how the mediators of DNA methylation, the DNA methyltransferases, are regulated at the level of the protein in response to the stimuli of the media. The differential makeup of the protein complexes of Dnmt1, referred to as the main DNA methyltransferase, and of its obligate co-factor, Uhrf1, were characterised in a proteomic screen. This led to the identification of several candidates and their potential roles in mediating DNA methylation have been postulated to build a case for future investigation. Furthermore, the influence of citrullination, a relatively understudied post-translational modification detected throughout early development, was also investigated in these model culture systems.

Understanding stem cell behaviour and the mechanisms that govern stem cell proliferation is a key question in developmental biology. Stem cell divisions must be carefully regulated as too little proliferation can cause developmental defects or reduce the response to injury, whilst too much proliferation can induce overgrowth. In adult mammalian tissues many stem cell populations reside in a quiescent state, in which the cell cycle is arrested and divisions cease. When needed, these stem cell can resume divisions in response to signals from the environment, a process known as reactivation. In this way stem cells can balance the amount of proliferation in order to maintain tissue homeostasis. During the larval development of Drosophila melanogaster, neural stem cells, also known as neuroblasts, undergo a similar period of quiescence. Neuroblasts divide during embryogenesis to generate neurons and glia for the larval nervous system. Just before hatching neuroblasts enter quiescence and cease diving. They also adopt a specific morphology in which a long primary process is extended centrifugally into the centre of the brain. As the larvae eat, the intake of amino acids signals to glia at the surface of the brain to release insulin like peptides that are sensed by neuroblasts and signal for reactivation to occur. Neuroblasts then retract their primary processes, round up and resume divisions to generate a second wave of neurons and glia which make up the adult central nervous system. Whilst insulin signalling is known to be involved in this reactivation switch, it remains unclear as to whether this is the only signalling pathway involved. Moreover, the primary process that is extended during quiescence is directed away from the glial insulin signal, and towards the synapse-dense neuropil in the centre of the brain. In this thesis, I investigate the role of these neuroblast primary processes and their effect on stem cell behaviour, including quiescence and reactivation. I have found that quiescent neuroblasts can be subdivided into four distinct groups based on the locations in which their primary processes terminate and the cell types that they contact. Distinct neuroblast lineages consistently target their processes to specific regions of the neuropil and this targeting is maintained throughout quiescence. Neuroblasts were found to frequently contact each other via their processes and direct cell-cell contacts were found between processes and both neurons and glia in the neuropil. This work identified several novel interactions between neuroblasts and their niche, which occur via their primary processes. In order to assess the function of the primary process several methods of disruption were tested, and multiphoton ablation was identified as a promising technique to physically disrupt process maintenance. Furthermore, the functional significance of neuroblast-neuroblast, neuroblast-neuron and neuroblast-glia interactions was investigated, and I have identified neuropil glia as important regulators of neuroblast reactivation. These data offer an insight into the role of the neuroblast primary process, a distinct morphological feature whose function was previously unknown.

High turnover in the haematopoietic system is sustained by stem and progenitor cells, which divide and mature to produce the range of cell types present in the blood. This complex system has long served as a model of differentiation in adult stem cell systems and its study has important clinical relevance. Maintaining a healthy blood system requires regulation of haematopoietic cell fate decisions, with severe dysregulation of these fate choices observed in diseases such as leukaemia. As transcriptional regulation is known to play a role in this regulation, the gene expression of many haematopoietic progenitors has been measured. However, many of the classic populations are actually extremely heterogeneous in both expression and function, highlighting the need for characterising the haematopoietic progenitor compartment at the level of individual cells. The first aim of this work was to chart the single-cell transcriptional landscape of the haematopoietic stem and progenitor cell (HSPC) compartment. To build a comprehensive map of this landscape, 1,654 HSPCs from mouse bone marrow were profiled using single-cell RNA-sequencing. Analysis of these data generated a useful resource, and reconstructed changes in gene expression, cell cycle and RNA content along differentiation trajectories to three blood lineages. To investigate how single-cell gene expression can be used to learn about regulatory relationships, data measuring the expression of 41 genes (including 31 transcription factors) in 2,167 stem and progenitor cells were used to construct Boolean gene regulatory network models describing the regulation of differentiation from stem cells to two different progenitor populations. The inferred relationships revealed positive regulation of Nfe2 and Cbfa2t3h by Gata2 that was unique to differentiation towards megakaryocyte-erythroid progenitors, which was subsequently experimentally validated. The next study focused on investigating the link between transcriptional and functional heterogeneity within blood progenitor populations. Single-cell profiles of human cord blood progenitors revealed a continuum of lympho-myeloid gene expression. Culture assays performed to assess the functional output of single cells found both unilineage and bilineage output and, by investigating the link between surface marker expression and function, a new sorting strategy was devised that was able to enrich for function within conventional lympho-myeloid progenitor sorting gates. The final project aimed to study changes to the HSPC compartment in a perturbed state. A droplet-based single-cell RNA-sequencing dataset of 44,802 cells was analysed to identify entry points to eight blood lineages and to characterise gene expression changes in this transcriptional landscape. Mapping single-cell data from W41/W41 Kit mutant mice highlighted quantitative shifts in progenitor populations such as a reduction in mast cell progenitors and an increase towards more mature progenitors along the erythroid trajectory. Differential gene expression identified upregulation of stress response and a reduction of apoptosis during erythropoiesis as potential compensatory mechanisms in the Kit mutant progenitors. Together this body of work characterises the HSPC compartment at single-cell level and provides methods for how single-cell data can be used to discover regulatory relationships, link expression heterogeneity to function, and investigate changes in the transcriptional landscape in a perturbed environment.

All cancers were once normal cells. They became cancerous through the chance acquisition of particular somatic mutations that gave them a selective advantage over their neighbours. Thus, the mutations that initiate cancer occur in normal cells, and the normal clonal dynamics of the tissue determine a mutant cell's ability to establish a malignant clone; yet these remain poorly understood in humans. One tissue was selected for the exploration of each of these two facets of somatic evolution: blood for clonal dynamics; colon for mutational processes. Blood presents an opportunity to study normal human clonal dynamics, as clones mix spatially and longitudinal samples can be taken. We isolated 140 single haematopoietic stem and progenitor cells from a healthy 59 year-old and grew them in vitro into colonies that were whole genome sequenced. Population genetics approaches were applied to this dataset, allowing us to elucidate for the first time the number of active haematopoietic stem cells, the rate at which clones grow and shrink, and the cellular output of stem cell clones. Colonic epithelium is organised into crypts, at the base of which sit a small number of stem cells. All cells in a crypt ultimately share an ancestor in one stem cell that existed recently, and consequently share the mutations that were present in this ancestor. We exploited this natural clonal unit, isolating single colonic crypts through laser capture microdissection. 570 colonic crypts from 42 individuals were whole genome sequenced. We describe the burden and pattern of somatic mutations in these genomes and their variability across and within different people, identifying some mutational processes that are ubiquitous and others that are sporadic. Targeted sequencing of an additional 1,500 crypts allowed us to quantify the frequency of driver mutations in normal human colon. Together, these two studies inform on the somatic evolution of normal tissues, describing new biology in human tissue homeostasis and providing a window into the processes that govern cancer incidence.

Deeper understanding of the embryological origins of tissues and organs is likely to provide insights into novel clinically relevant preventative and therapeutic strategies. To do so effectively and at a large scale so as to have clinical significance requires an exhaustive and meticulously accurate knowledge of normal morphological development and the underlying molecular pathways. A variety of lineage tracing and classical molecular biology techniques have led to key insights but emerging technologies now offer the possibility of more fine grained and precise measurements at the level of the single cell rather than ensembles of heterogeneous cells. In this study the rapidly developing technology of single cell RNA sequencing was combined with the development of state of the art computational methods to study murine gastrulation and early organogenesis at a hitherto unprecedented granularity. A comprehensive analysis of FLK1+ cells harvested from gastrulating murine embryos was performed. In-silico reconstruction of pseudo-temporal and pseudo-spacial relations were demonstrated. Using prior knowledge of known driver genes deeper substructure was revealed in clusters that were initially defined by unsupervised algorithms, illustrating that careful implementation of supervised approaches can outperform naïve unsupervised methods. In this way multiple members of the leukotriene branch of the arachidonic acid pathway were found to be enriched within a subset of the endothelial cluster that has a molecular signature consistent with yolk sac derived definitive wave haematopoiesis. This was subsequently validated in an in-vitro embryonic stem cell differentiation colony assay. By developing a novel adaptation to tSNE dimensionality reduction that now allows new data points to be mapped backed to previously calculated embeddings, the FLK1+ data set became a reference to which cells from other experiments were mapped. The Tal1-/- knockout mutant was characterised, reaffirming the known phenotype with complete failure of embryonic haematopoiesis. Analysing the Tal1-/- endothelial cluster shows definitively in the in-vivo organism no activation of an alternative cardiac fate programme as was previously postulated from an in-vitro model system. Additionally tSNE mapped Brachyury cells into the void in the FLK1+ dataset and identified a node like population. Loss of spacial context remains an Achilles' heel of single cell protocols. Generation of the single cell suspensions leads to disruption of cellular contacts and loss of any spacial information. A novel method is described which uses tSNE of bulk spacial data to pre- condition a tSNE map upon which single cells can then be computationally positioned reconstructing spacial context. To model gene interactions and perturbations from single-cell data, a hybrid feed-forward deep neural network was trained on branching pseudo-temporally arranged single cell qPCR data of in-vivo wild-type murine developmental haematopoiesis. Strikingly despite the model never having 'seen' a mutant, in-silico gene perturbations in the deep neural network are able to faithfully reproduce the Tal1-/- phenotype. In summary the use of single cell transcriptomics to probe early murine embryology com- bined with development of new methods has uncovered a novel pathway in embryonic haematopoietic development and allowed in-silico reconstruction of a short period of early embryonic haematopoiesis. Critically these methods have broad application within the fields of developmental and stem cell biology.

In this thesis I set out to answer the question 'is differentiation robust to replication impediments?'. Prior work in the group has focussed on the epigenetic impact of replication impediments in the terminally differentiated DT40 chicken cell line. I wanted to find out whether these impediments could impact the fundamental biological process of embryonic development. In this work the BOBSC human induced pluripotent stem cell line was differentiated to definitive endoderm while replication was perturbed using DNA damaging agents, low dose hydroxyurea and G-quadruplex secondary structure stabilisation. DNA damage was induced both in the undifferentiated state and during differentiation. When damage was induced during differentiation, greater levels of cell death were seen and there was a larger increase in cells in G2/M phase of the cell cycle compared with treatment in the undifferentiated state. The efficiency of differentiation was observed to negatively correlate with the dose of DNA damaging agent. During unperturbed differentiation, the levels of DNA damage response proteins, including p53, were found to decrease. However, when DNA damage was induced during differentiation the level of p53 increased. In order to understand whether the upregulation of p53 was preventing differentiation, TP53-/- cells were differentiated in the presence of DNA damaging agents. These cells were found to differentiate as efficiently as wildtype untreated cells. This indicates that p53 prevents differentiation in the presence of DNA damage, termed here a 'differentiation checkpoint'. The level of γH2A.X was found to be markedly increased 50 hours into unperturbed differentiation. This correlates with the point at which the cells transition from being epithelial to mesenchymal. The role of this rise is not known but may correspond to massive transcriptional changes that occur during this transition. G-quadruplex-binding ligands were also shown to alter the course of differentiation, either by a p53-dependent mechanism or by a separate, possibly G-quadruplex specific, mechanism. This also occurred in REV1-/- cells, a specialised polymerase known to play a role in G-quadruplex-processing. This work has led to the conclusion that replication impediments are able to alter the course of definitive endoderm differentiation in human induced pluripotent stem cells.

Background The continuously growing mouse incisor provides a robust model for studying molecular mechanisms of stem cell (SC) fate determination. While the epithelial SCs are well studied within this model, the identification and characterisation of a mesenchymal SC (MSC) population has yet to be defined. Aims This study aims to identify the molecular signatures of a novel MSC pool within the lower murine incisor. This work aims to investigate the molecular mechanisms governing the maintenance and transition of MSC cells and their progeny, particularly the role of Notch signalling in this process. Methods Isolation of proposed putative MSC containing and known mesenchymal transit amplifying cell (MTAC) containing regions were undertaken. Laser capture microdissection and subsequent comparative analysis of MSC marker expression between populations was performed. Investigation of the molecular mechanisms governing MSC activation and maintenance was undertaken. Specifically, the role of Notch signalling was investigated, through analysis of Notch pathway transgenic mouse models including tissue specific RBP-Jκ knock out, Dlk1 null, and tissue specific Dlk1 overexpressing strains. In vitro models were developed to validate findings. Results MTAC and MSC containing regions exhibited distinct expression signatures of quiescent MSC marker genes. Notch pathway ligands, receptors and downstream effectors were differentially expressed between these populations. Modulation of Notch signalling in vivo impacted the behaviour of incisor MSCs. Manipulation of Dlk1 in vitro identified it as a potent regulator of MTAC maintenance. Conclusions The novel endogenous MSC population exists within the mouse incisor mesenchyme. The MSCs give rise to cells of the MTAC region, which in turn express Notch ligand Dlk1. Dlk1 is pivotal in balancing the lineage differentiation and maintenance of the incisor MSCs. Thus, Notch signalling plays a key role in the molecular regulation of the activation of tooth mesenchymal stem cells.

Since its discovery, CD133 has been used to identify, isolate and characterise many different stem cell types across a wide variety of tissues. Nonetheless, to date not much has been elucidated about its function in these cells. The continuously growing mouse incisor contains a large population of epithelial stem cells that drive the constant need for enamel generation. Using this model, this project aims to elucidate the role of CD133 in epithelial stem cell renewal and activation. After validating the presence of CD133 in the developing mouse incisor epithelium, this project compared the dental epithelial stem cell dynamics of wildtype mice versus CD133 KO mice. Mutant mice displayed significant anomalies across the whole dental epithelium, ultimately, leading to defects in the enamel, the product of fully differentiated ameloblasts. Subcellular analysis of the dental incisor epithelium in CD133 KO mice revealed disturbed primary cilia homeostasis. Primary cilia are crucial in interpreting extracellular cues and CD133 KO primary cilia specifically fail to transmit Shh-mediated signals. Further investigation of the functions of CD133 revealed that CD133 is important for the sequential recruitment of Arl13b and Hdac6 to the primary cilium regulating proper ciliogenesis. Establishment of an in vitro cell culture model of dental epithelial cells allowed the investigation of nuclear CD133 under normal physiological conditions. To date, nuclear CD133 has been reported in a variety of cancers. This study provides insight into the formation of the CD133-Glis2 complex in the nucleus. This complex regulates the expression of Stat3, an important regulator of stem cells, in the dental epithelium. In summary, this study has elucidated the importance of CD133 on primary cilium homeostasis, which in turn regulates stem cell renewal and activation in the developing incisor epithelium.

Liver injury stimulates hepatocyte proliferation, regenerating the liver through self-replication. In cases where there is severe, repetitive, parenchymal damage, as seen in human chronic liver disease, hepatocyte mediated regeneration becomes impaired. In this setting it is currently unclear whether endogenous biliary epithelial cells can repopulate the hepatocyte compartment. This thesis therefore aimed to address this point by lineage tracing the main two liver epithelia populations on a background of impaired hepatocyte regeneration. To impair regeneration, an Itgb1 transgene was specifically deleted, conditionally, from the hepatocyte epithelium. Long-term loss of β1-Integrin alone or with additional injury caused an epithelial ductular reaction of biliary origin. Alongside β1-Integrin ablation, the hepatocyte epithelium was also labelled with a heritable ROSA26LSLtdTomato reporter. Impaired hepatocyte regeneration mediated by β1- integrin ablation resulted in 25% of hepatocytes becoming tdTomato negative (non-hepatocyte derived). To verify that the non-hepatocyte mediated regeneration was originating from the biliary epithelium, anti-Itgb1 RNAi was administrated to K19CreERT LSLtdTomato mice. Resulting in tdTomato positive hepatocytes that had differentiated from the labelled tdTomato positive biliary epithelial cells. In summary, this thesis demonstrates that hepatocyte β1-Integrin ablation combined with toxic damage causes marked ductular reactions and results in a substantial regeneration of functional hepatocytes from the biliary epithelium.

The intestinal epithelium is rapidly renewing throughout life. A population of stem cells exist within the intestinal crypt that drive rapid cell renewal and replace each other by a pattern of neutral drift. Perturbation of these dynamics through oncogenic mutation can predispose the epithelium to neoplastic transformation. Understanding the factors that govern these dynamics will give insight into the early stages of oncogenesis. Continuous clonal labelling, whereby DNA strand slippage leading to the contraction or expansion of a microsatellite during mitotic replication, can be employed to enable the detection of a single clone. Previous studies have shown that quantification of clone size over time allows inference of the functional stem cell number and stem cell replacement rate within intestinal crypts. Current continuous labelling techniques require the introduction of a transgenic microsatellite into a model system genome, such as mouse, that leads to reporter expression following mutation. This obviously precludes human studies. Alternative somatic alterations techniques used for continuous labelling in humans requires spontaneous loss of a protein, or change in methylation status, within a single clone that can act as a clonal mark. Though these techniques have given insight into the spread of mutations within the intestinal epithelium and enabled inference of adenoma clonality, the true neutrality of these changes are currently unknown. We propose that the small changes in endogenous microsatellite length will act as a neutral clonal mark within the intestinal epithelium and allow an unbiased approach to quantifying intestinal stem cell dynamics in human intestinal tissues. To overcome the many technical challenges associated with accurate measurement of microsatellite length, a stepwise approach was taken to develop a technique for the multiplexed high throughput sequencing of up to 21 native dinucleotide repeats in hundreds of single crypts. Furthermore, a novel method was developed for the quantification of clone size from data generated from the targeted re-sequencing of microsatellites in single crypts. This protocol was validated in vitro and in vivo in mouse. Furthermore, proof of principle sequencing in human crypts was performed to show that this method is suitable for larger scale quantification of intra-cryptal clone size in human tissue. This, and similar approaches, may be the only way to quantify intestinal stem cell dynamics within the healthy human colon or, dysplastic or adenomatous patient tissue. These measurements should give a unique insight into the dynamics of healthy, pre-neoplastic and neoplastic human intestinal stem cells.

While a large body of work has focused on the transcriptional regulation of cellular identity, the role of the mechanical properties of cells and the importance of their physical interactions with the local environment remains less well understood. In this project, we explored the impact of cytoskeleton-generated forces exerted on the nucleus in the context of early embryonic stem (ES) cell fate decisions. We chose to perturb force generating components in the cytoskeleton – notably the molecular motor non-muscle myosin II - and key structural and chromatin binding proteins in the nuclear envelope, notably, the lamins (LMNA), Lamin B receptor (LBR) and components of the LINC complex (nesprins/KASH). The structural proteins in the nuclear envelope regulate both the mechanical response of the nucleus to force and the stabilization of peripheral heterochromatin (repressed genes). Our hypothesis is that reducing forces transmitted directly to chromatin or increasing tethering of peripheral heterochromatin to the nuclear envelope would restrict access to lineage specific genes sequestered at the nuclear lamina and thereby either impair, or delay, differentiation. We found phenotypes in the capacity of mouse ES cells to specify to the neural lineage following our perturbations: overexpression of LMNA, LBR and KASH proteins resulted in a significant fraction of cells that did not express the neuroectoderm marker Sox1 after four days of differentiation, while inhibiting non-muscle myosin II delayed Sox1 expression in the entire population. Overexpression of LMNA and LBR did not affect the ability of the cells to exit the naive pluripotent state, which raises the possibility that the perturbations are halting the cells in a formative phase prior to lineage specification. Future work will focus on looking at genome-wide transcriptional changes accompanying differentiation combined with an analysis of spatial information of differentially regulated genes.

Haematopoietic stem cells (HSCs) are capable of differentiation into all mature haematopoietic lineages, as well as long-term self-renewal and are consequently able to sustain the adult haematopoietic system throughout life. Currently, in the mouse, HSCs are understood to first appear in the aorta-gonad-mesonephros (AGM) region at embryonic day 11 via a process of maturation from precursors (pre-HSCs). This maturation within the AGM region involves the complex interplay of signalling between cells of the niche and maturing precursor cell populations, but is relatively little understood at a molecular level. Recently our understanding of the AGM region has been refined, identifying the progression from E9.5 to E10.5 and the polarity along the dorso-ventral axis as clear demarcations of the supportive environment for HSC maturation. In this thesis, I investigated the molecular characteristics of these spatio-temporal transitions in the AGM region through the application of RNA-sequencing. This enabled the identification of molecular signatures which may underlie the supportive functionality of the niche. I further compared these expression signatures to the transcriptional profile of an independent cell type, also capable of supporting HSC maturation, the OP9 stromal cell line. By combining this transcriptional information with an ex vivo culture system, I screened a number of molecules for their ability to support HSC maturation from early precursors, leading to the discovery of a novel regulator of HSC maturation: BMPER. Further characterisation of this molecule enabled the identification of its specific cellular source and the proposal that through its action as an inhibitor of BMP signalling it facilitates the maturation of precursors into HSCs. These results lend further detail and support to the role of BMP signalling in the regulation of HSC maturation as well as demonstrating the potential of these transcriptional profiles to yield novel mechanistic insight.

Breast cancer is the most common cancer in women and unspayed female dogs. The Epithelial to Mesenchymal Transition (EMT) is a process involved in embryogenesis, carcinogenesis, and metastasis. The Transforming Growth Factor- Beta (TGF-β) pathway and its associated transcription factors are crucial for EMT induction, during which epithelial cells lose their defining characteristics and acquire mesenchymal properties. EMT has been implicated as a driver of metastasis as it allows cells to migrate and invade different organs. Recent evidence indicates that cancer stem cells are required to establish metastatic tumours at distant sites, and that EMT may promote development of cancer cells with stem-cell characteristics, thus, the EMT pathway may be an important molecular determinant of tumour metastasis. The main objective of this project was to characterise TGF-β-induced EMT in breast cancer models. EMT was induced by TGF-β in human, canine and feline breast cancer cell lines, and confirmed by morphological changes and molecular changes at the protein level by Western blot analysis. Changes at the mRNA level were confirmed in human and canine mammary carcinoma cell lines by qRT-PCR; migratory properties were assessed by invasion assays in vitro in feline and canine mammary carcinoma cells. Importantly, we observed that feline and canine mammary carcinoma cells stimulated by TGF-β acquired stem cell characteristics including sphere-forming ability, self-renewal, and resistance to apoptosis, and also enhanced migration potential. Canine cells showed resistance to chemotherapeutic drugs after TGF-β stimulation. These data suggests a link between EMT and cancer stem-cells. Moreover, global changes in microRNA expression were mapped during TGF-β-induced EMT of canine mammary carcinoma cells. This gave significant insight into the regulation of EMT in canine cancer cells and identified several potential targets, which require further investigation. During EMT cells acquire migratory properties and cancer stem-cell characteristics, suggesting that EMT and the stem-cell phenotype are closely related during cell migration and metastasis, therefore making the TGF-β pathway a potential target for the development of novel therapies against cancer and its progression.

The exogenous expression of master transcription factors (TFs) to drive cell identity changes is an exciting and powerful approach to cell and tissue engineering. Yet, the generation of desired cell types is often plagued by inefficiency and inability to produce mature cell types. Through investigations of the molecular mechanisms of induced pluripotent stem cell (iPSC) generation, I discovered that expression of constitutively active Smad2/3 (Smad2CA/3CA), together with the Yamanaka factors, could dramatically improve the efficiency of reprogramming. Mechanistically, SMAD3 interacted with both co-activators and reprogramming factors, bridging their interaction during reprogramming. Because SMAD2/3 interact with a multitude of master TFs in different cell types, I tested the conversions of B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons. Remarkably, each conversion system was markedly enhanced when the master TFs were co-expressed with Smad3CA. These results revealed the existence of shared molecular mechanisms underlying diverse TF-mediated cellular conversions, and demonstrated SMAD2/3 as a widely applicable cofactor that potentiates the generation of diverse cell types with profound efficiency and maturity.

The behaviour of individual cells must be carefully coordinated across a tissue to achieve correct function. In particular, proliferation and differentiation decisions must be precisely regulated throughout development, tissue maintenance, and repair. A better understanding of how these processes are controlled would have implications for human health; cancer is, after all, dysregulated proliferation, while regenerative medicine relies on being able to influence cell decisions accurately. To investigate such fundamental biological processes, it is common practice to use an experimentally tractable model organism. Here, we focus on the germ line of the nematode worm C. elegans, which provides opportunities to study organogenesis, tissue maintenance, and ageing effects. Despite the advantages of this organism as a biological model, certain questions about germ cell behaviour and coordination remain challenging to address in the lab. There is therefore a need for computational models of the germ line to complement experimental approaches. In this thesis, we develop a new in silico model of the C. elegans germ line. Novel aspects include working in three dimensions, covering the late larval period, and integrating a logical model of germ cell behaviour into a wider cell mechanics simulation. Our model produces a reasonable fit to wild-type germline behaviour, and provides the first cell tracking and labelling predictions for the larval period. It also suggests two new biological hypotheses: 1) that “stretching” growth plays a significant role in gonadogenesis, and 2) that a feedback mechanism acts on the germ cell cycle to prevent overproliferation. Having introduced the full model, we address some technical questions arising from our work, namely: what is the effect of applying a more physically realistic force law?; and can simulation performance be improved by changing the numerical scheme? Finally, we use in silico modelling to compare a number of hypothesised germ line maintenance mechanisms. There, our results support a model with functionally equivalent germ cells undergoing at most infrequent, transient cell cycle arrests.

Prostate cancer is a leading cause of cancer-related death in men, and while many men diagnosed with the disease will have an indolent clinical course, 20-25% of men will experience disease recurrence which is invariably lethal. There is an urgent need for prognostic biomarkers that will predict disease recurrence and risk-stratify patients upon diagnosis, allowing for personalised therapies. This thesis attempts to identify new prognostic biomarkers for prostate cancer and investigates their patterns of protein expression in human primary prostate tumour tissue. Cancer stem cells are cancer cells thought to be uniquely capable of self-renewal and tumorigenicity, and may have a role in tumour recurrence. Using a literature searching approach, potential biomarkers related to stem cells, cancer stem cells or recurrence in prostate cancer were identified, and ALDH7A1, BMI1, SDC1, MUC1-C, Nestin and ZSCAN4 were chosen for investigation. An in silico approach was also used for biomarker identification, with RS1 and SLC31A1 selected as their mRNA was found to be upregulated in recurrent tumours. The expression patterns of all 7 potential biomarkers were examined by immunohistochemistry on prostate tumour tissue and benign tissue from prostate biopsies and prostatectomies. BMI1, ALDH7A1, MUC1-C and Nestin showed no relationship to recurrence or other clinical features. RS1 protein levels increased in patients with recurrence within 5 years, negatively correlated with AR expression, and a meta-analysis showed that the RS1 gene was amplified in up to 32% of castration-resistant prostate tumours. ZSCAN4 was heterogeneously expressed in a subset of 26% of prostate tumours with unclear characteristics and was not expressed in benign tissue, but was not associated with recurrence. Finally, SDC1 expression was lost in tumour epithelium, but a population of unidentified SDC1-expressing cells were found in the stroma of a third of tumours, and an increased burden of these cells was associated with primary Gleason pattern 5 tumours. These cells do not overlap with common epithelial, mesenchymal or stromal lineages, but may be migratory. In summary, the data presented in this thesis identifies 3 potential new biomarkers for prostate cancer, and provides the basis for future characterisation of their wider roles in the disease.

Background: Intrahepatic cholangiocarcinoma (CCA) is a highly treatment-resistant malignancy of biliary epithelium with increasing global mortality. Histologically, CCA is characterised by a pronounced inflammatory stroma of tumour-associated myofibroblasts, macrophages, immune cells and a modified extracellular matrix (ECM). In other solid cancers, the stroma plays a tumour promoting role. The functional role of the stroma in CCA remains unclear. The origin and the proportional contribution to the stroma by haematopoietic and mesenchymal bone marrow (BM) -derived cells is not known in CCA. Intriguingly, reports suggest that mesenchymal stem cells (MSCs) may contribute to the epithelial compartment of malignant tumours. Furthermore, the Notch signalling pathway is known to play oncogenic and tumour suppressive roles in diverse neoplasms but its role in CCA remains unclear. Aims and Methods: The functional role of myofibroblasts and macrophages in the tumour stroma of CCA was investigated together with an analysis of the origin and contribution of BM-derived cells to the stromal and epithelial compartments of CCA. The Notch signalling pathway was studied as a potential signalling mechanism through which the stroma and malignant epithelial compartments of CCA may interact. Results: The thioacetamide rat model of CCA was optimised and found to display excellent histological congruence with human lesions. The tumour cellular microenvironment comprised of myofibroblasts, migratory macrophages and immune cells. During cholangiocarcinogenesis, progressive intrahepatic accumulation of inflammatory cells and proliferation of bipotential progenitor cells preceded the development of invasive CCA. In vitro, CCA lines were identified to contain a side population of stem cells. Adoptive transfer of BM from Enhanced Green Fluorescent Protein (EGFP) transgenic rats to wild type rats to establish chimeras was undertaken. In transplanted rats, persistent EGFP+ chimerism of both haematopoietic and mesenchymal stem cell compartments was established. In tumours, macrophages and neutrophils were overwhelmingly EGFP+ve, whereas myofibroblasts, fibroblasts and benign and malignant bile ducts were EGFP-ve. There was no evidence of cell fusion or EGFP silencing. These findings were confirmed in spontaneous breast, skin and colon tumours in EGFP+ chimeric rats not treated with TAA. In vitro studies to recapitulate the cellular and extracellular elements of the tumour niche identified that ECM components induce characteristic cell proliferation patterns dependent on the matrix component but do not appear to affect chemosensitivity. Bidirectional interaction between CCA cells and hepatic stellate cells (mediated by soluble factors) was identified. Furthermore, in direct co-culture, M2 polarised macrophages appear to enhance CCA cell proliferation compared to M1 macrophages. In considering the Notch pathway, Notch signalling components (particularly Notch3) and target genes were upregulated in human CCA specimens. Immunohistochemical analysis identified apparent distribution of Notch ligand on tumour stroma and Notch receptor subtypes on malignant epithelia. Although direct co-culture of CCA cells with myofibroblasts and M1/M2 polarised macrophages did not clearly demonstrate stromal:epithelial Notch pathway activation, this may have been a function of in vitro experimental limitations. Gamma-secretase inhibition downregulated the Notch pathway, reduced proliferation and appeared to enhance chemosensitivity of CCA cells in vitro. Conclusions: A stereotypical niche forms around CCA in developing and malignant lesions. There was no evidence of a BM-derived stem cell contribution to the epithelial component of CCA, breast, colon or skin malignancies. Haematopoietic but not mesenchymal components of the tumour stroma were of BM origin. Notch signalling is upregulated in CCA and appears to play a tumour promoting role in CCA; pathway inhibition represents a potential therapeutic target.

Tissue engineers – in order to develop therapies for the treatment of complex neurological injuries and diseases – attempt to recreate elaborate developmental mechanisms in vitro. Neuronal precursor cells are excellent candidates for the study of developmental operations such as cell adhesion, differentiation, and axonal pathfinding. Hyaluronan (HA) is a common polysaccharide that is found extensively throughout the neuronal extracellular matrix (ECM), and can be functionalised and crosslinked to form stable hydrogels that support growing neuronal cells. Hyaluronan hydrogels can be modified chemically and mechanically to mimic the ECM of the developing brain, awarding control over mechanisms such as differentiation and axonal pathfinding. This thesis is concerned with the functionalisation and characterisation of HA hydrogels, ultimately in order to simulate vital properties of the developing brain. Here we show that HA hydrogels can be finely tuned mechanically (by modulating stiffness and viscosity), and chemically, by the conjugation of peptides that mimic the neural cell adhesion molecule (NCAM). NCAM mimics and novel mimics of sialylated NCAM significantly influence the differentiation of NSPCs in 2D and 3D. HA hydrogels successfully support long term culture of neural cells in 3D, and encourage the formation and extension of neurites of several cell types including human, mouse and rat neuronal precursor and stem cells. These results demonstrate for the first time that novel NCAM mimicking peptides can be conjugated to well defined hydrogel matrices that influence intricate developmental behaviours in 3D. Understanding how neural cells form functional networks is essential for the development of clinical approaches that attempt to address the injuries and diseases that affect these systems.

The haematopoietic stem cell (HSC) is found in the adult human bone marrow, where it gives rise to all the circulating blood cells throughout adulthood. Understanding the signalling events that programme these cells during development will improve HSC in vitro culture, their generation from embryonic stem cells or induced pluripotent stem cells, and their potential therapeutic application. HSCs bud from the floor of the dorsal aorta and seed the bone marrow via circulation. The precursors to the dorsal aorta and HSCs are called haemangioblasts, which are found in the dorsal lateral plate mesoderm in Xenopus. The knowledge of the location of these precursors allows their programming to be studied in detail during embryonic development. A key pathway implicated in the programming of HSCs is the BMP signalling pathway. Here, using both a small molecule inhibitor and a transgenic Xenopus line, BMP signalling has been inhibited post-gastrulation without perturbing the gross morphology of the embryo. This has shown that BMP signalling is required for HSC programming in the dorsal lateral plate mesoderm via the expression of a critical haematopoietic transcription factor, gata2. Morpholino knockdown of evi3has revealed it to be essential for HSC programming in the dorsal lateral plate mesoderm, where it is required for the expression of gata2. Furthermore, as evi3 is known to bind to the active BMP signalling complex, and as evi3 knockdown phenocopies post-gastrulation BMP inhibition, evi3 appears to be required for BMP signalling to initiate gata2 expression in the DLP. Taken together, the findings presented here demonstrate an essential post-gastrulation role of BMP signalling and Evi3 for programming HSCs in Xenopus.

One strategy to restore vision in retinitis pigmentosa and related retinal degenerations is by cell replacement. Typically, patients lose vision when the outer retinal photoreceptor layer is lost, and so the therapeutic ideal would be to restore vision at this stage of disease. It is not currently known if a degenerate retina lacking the outer nuclear layer of photoreceptor cells would allow the survival, maturation and reconnection of replacement photoreceptors, as prior studies used hosts with a pre-existing outer nuclear layer at the time of treatment. Here, using a murine model of severe human retinitis pigmentosa at a stage when no host rod cells remain, transplanted rod precursors are shown to reform an anatomically distinct and appropriately polarised outer nuclear layer. A trilaminar nuclear organisation is returned to the rd1 hosts that had only two retinal layers before treatment. The newly introduced rod precursor cells were able to resume their developmental programme in the degenerate host niche to become mature rods with light- sensitive outer segments, and reconnected with host neurons downstream. Visual function, assayed in the same animals before and after transplantation, was restored in animals with zero rod function at baseline. These observations suggest that a cell therapy approach may reconstitute a light-sensitive cell layer de novo and hence repair a structurally damaged visual circuit. Rather than placing discrete photoreceptors amongst pre-existing host outer retinal cells, total photoreceptor layer reconstruction may provide a clinically relevant model to investigate cell-based strategies for retinal repair.

Cardiac and orthopaedic diseases are significant causes of morbidity and mortality in dogs and are therefore critical areas for veterinary research. More information regarding the pathophysiology of these diseases, and the development of novel therapeutics are sorely required and adult stem cells (ASCs) are a promising source of cells for both investigation of these diseases in vitro and also potentially therapeutics in the longer term. ASCs are a readily available source of multipotent cells which bypass the ethical issues surrounding embryonic stem (ES) cells. ASCs have been described in several tissues of the body, and typically differentiate along specific cellular routes related to original source location. This thesis investigates whether ASCs can be isolated and cultured from the dog from two specific locations; cardiac, producing cardiac stem cells (CSCs); and the bone marrow, producing mesenchymal stem cells (MSCs). These cell sources will be extensively characterised at their baseline for morphology, culture behaviour and gene marker expression. Following characterisation each cell source will be subjected to differentiation techniques to examine canine ASC multipotent differentiation potential. CSCs were isolated from cultured atrial cardiac explant tissue taken from dogs post-mortem, with owners’ consent. These cells were able to survive successive passages in serum free media and formed large spherical cell clusters, termed ‘cardiospheres’. CSCs were capable of clonal expansion under controlled culture conditions, demonstrating their ability for self-renewal. Characterisation of these cells demonstrated the expression of CSC markers; c-Kit, GATA 4 and Flk-1 and no expression of cardiac lineage markers including cardiac troponin T and I, Nkx2.5, the cardiac ryanodine receptor and the β1-adrenergic receptor. Primary canine MSCs were isolated from bone marrow aspirates using ficoll separation and cultured on tissue culture plastic. Canine MSCs closely resembled MSCs described from other species, such as the human and mouse, and were found to express CD44 and STRO-1 and were negative for CD34 and CD45. CSCs and MSCs were exposed to published cardiac directed differentiation protocols and differentiation then analysed using cellular morphology and gene expression. Canine CSCs appeared to differentiate partially along cardiac lineages with upregulation of cardiac troponin T and Nkx2.5, and down regulation of c-Kit and endothelial lineage markers. Canine MSCs demonstrated some morphological changes during cardiac differentiation, and demonstrated up-regulation of Nkx2.5 and Flk-1 but no significant alteration in other markers examined. This suggested that cardiac directed differentiation was not as successful with canine MSCs compared to CSCs and conflicting with published data using rodent MSC models. Murine MSCs were used as a positive control cell line for cardiac directed differentiation, based upon published literature. Critically there were key marker expression differences between baseline murine and canine MSCs, including the expression of cardiac markers such as cardiac troponin T and I, and the Ryanodine receptor. Furthermore, expression analysis of cardiac genes changed with time in culture and passage number and no significant alteration was seen when cells were subjected to the cardiac differentiation protocol; thereby bringing into question the data regarding successful cardiac differentiation using murine MSCs. Canine MSCs were further differentiated toward a chondrocyte lineage to investigate the use of MSCs for orthopaedic research. Canine MSCs were successfully differentiated toward articular type cartilage, with demonstration of extracellular matrix secretions, an upregulation of collagen type II with downregulation of collagen type I and the development of SOX9 expression in differentiated cells. This thesis builds the groundwork for future ASC research in the dog. Successful isolation and culture of two ASC sources from the dog is demonstrated. Cardiac and cartilage directed differentiation was successful using primary sourced cells, but differentiation was found to be limited to highly specific routes for each stem cell source. The results presented here highlight the importance of analysing baseline stem cells extensively prior to differentiation and in particular, before making comparisons between cell populations isolated from different locations and species.

Resistance to endocrine treatments in oestrogen receptor positive (ER+) breast cancer (BC) significantly contribute to patient morbidity and mortality. ER+ BC constitute 60% of all breast cancers although there is considerable clinico-pathological diversity within this group. Breast cancer initiating cells (BCICs) are implicated in tumour relapse and metastasis and are postulated to drive resistance to standard anti-cancer therapies. However little is known about the sensitivity of BCICs to endocrine therapies. We assessed the effect of tamoxifen treatment and acquired tamoxifen resistance on BCIC frequency in vitro and in vivo using breast cancer cell lines and, importantly, patient derived samples of early and metastatic ER+ breast cancer. In ER+ breast cancer, BCICs may be prospectively enriched in vitro by selecting cells by CD44+/CD24lo/ESA+ phenotype or by mammosphere initiating capacity (MIC). However the gold standard assay to determine BCIC frequency is limiting dilution transplantation in vivo. In the past it has been historically difficult to generate xenograft models of ER+ breast cancer using patient samples. In this thesis, using a novel experimental technique, patient-derived xenografts (PDX) of early and metastatic ER+ BC were generated with almost 85% efficiency in NOD/SCID IL2gammaR-/- (NSG) mice. PDX expressed ER and were able to undergo serial in vivo passage, matching the phenotype of the tumour from which they were derived. In this work, two patterns of response to tamoxifen treatment were observed in ER+ cell lines, patient derived breast cancer samples and xenografts during BCIC assays in vitro and in vivo; Limited Sensitivity (LS) or Resistance (R). In the LS group there was no change or a significant diminution in BCIC frequency in the presence of tamoxifen. In the R group, a significant increase in BCIC frequency was observed in the presence of tamoxifen. Furthermore BCIC activity was shown be enhanced by the acquisition of tamoxifen resistance using cell line models. Cellular populations enriched for BCICs in ER+ cell lines were shown to express low levels of ER compared to non-BCICs. Finally Notch (gamma-secretase inhibitor) and EGFR (gefitinib) pathway inhibitors were tested alone or in combination with tamoxifen against a panel of established and novel cell lines and ER+ patient-derived breast cancer samples for anti-BCIC activity. Tamoxifen treatment can increase BCIC frequency in vitro assays of cell lines and patient-derived samples and in vivo using patient-derived xenografts of ER+ breast cancer. However phenotypic diversity of BCIC may be present within the ER+ BC population. A pharmaceutical strategy to effectively treat BCICs alongside standard endocrine therapy is necessary for the effective future treatment of ER+ breast cancer.

Over the past decade the concept that the hair follicle plays an important role in cutaneous wound repair has been established. Several elegant lineage tracing studies have demonstrated that hair follicle derived cells contribute to the long term maintenance of the epidermis following repair, while an absence of hair follicles is known to delay repair. The exact mechanisms surrounding hair follicle derived repair are unknown. Moreover, while multiple stem cell niches are present within the hair follicle, their relative importance during wound repair is still unclear. The hair follicle is also a regenerative mini-organ, undergoing regular cycles of growth and regression throughout life, yet surprisingly this has not been previously investigated with respect to wound repair. Data presented in this thesis reveals an unappreciated, yet fundamental link between the independent processes of hair cycle and wound repair, with a substantial acceleration in the rate of repair (~50%) observed in anagen phase. Importantly, the hair follicle appears to play a global role in repair, with differences in the contribution of multiple cell types to wound repair. In addition, this thesis addresses the early kinetics of hair follicle wound response for the first time. Anagen hair follicles are found predisposed to a more rapid and extensive response to injury, suggesting a higher overall percentage of repair derived from the hair follicle in anagen phase. Surprisingly, the bulge stem cell region, while critical for hair cycle appears to play little role in the events immediately following injury, and is not required for initiation of re-epithelialisation. Gene expression profiling reveals numerous genes associated with anagen accelerated repair, and identifies altered modulation of the immune system as a key mechanism. Further, anagen wounds are associated with an upregulation of developmental transcription factors, which may imply a more regenerative healing phenotype. These data reveal numerous targets with the potential to accelerate repair, which now require validation for their therapeutic potential. These targets could be of importance in promoting the repair of chronic wounds, an area of unmet clinical need. More generally, this thesis has established hair cycle as an important experimental variable, which must be controlled for in all future in vivo murine wounding studies.

Cardiovascular disease is a leading cause of death worldwide and becomes increasingly prevalent in the elderly population. Independent of etiopathogenesis, heart failure (HF) is the final common stage of numerous heart diseases. Cardiac stem cell (CSC) therapy has emerged as a promising cell-based strategy for treatment of HF. However, cell replacement is not able to fully restore a structurally damaged myocardium in advanced and end-stage HF. The objective of this project was to test the following hypotheses: that a bioengineered heart extracellular matrix (ECM) with preserved intact geometric structure could be generated using decellularization by coronary perfusion; and that autologous CSCs, to repopulate this ECM, could be isolated and expanded from the adult heart, with the caveat that autologous CSCs are depleted and impaired by both aging and chronic dilated cardiomyopathy. This will help to develop a possible therapeutic approach for advanced HF, using a combination of CSCs and engineering technique. Resident CSCs were isolated from explant-derived cells (EDCs) and expanded into cardiosphere-derived cells (CDCs) via cardiosphere formation. The CDCs expressed CSC markers (c-kit and Sca-1), pluripotent markers (Oct3/4 and Sox2), and the cardiac lineage-committed marker (Nkx2.5), and showed clonal expansion, self-renewal, and cardiomyogenic potential in vitro. In tissue engineering experiments, CDCs survived and proliferated within biomaterial alginate scaffolds for up to 7 weeks. An engineered bioartificial ECM scaffold was successfully produced from a whole rat heart using retrograde coronary perfusion and possessed an intact 3D architecture with functionally perfusable vascular network. Compared with ventricles, cultures derived from atria produced significantly higher number of c-kit+ and Sca-1+ CSCs (c-kit: 13% vs. 3.4%; Sca-1: 82% vs. 53%, respectively) and exhibited greater clonogenic and proliferative capacity. CDCs could be grown from young and aged mice, but the yield of CSCs significantly declined with age, as did cell migration and differentiation potential. In comparison to wild-type mice, atrial-CDCs from dystrophic mice showed no significant differences in CSC subpopulations and characteristics, despite confirmation of cardiac dysfunction using MRI. In conclusion, CDCs could be considered to be a viable cell candidate for cardiac therapy and may be used to treat HF at various stages, in combination with myocardial tissue engineering.

Many countries in Latin America, for cultural, religious and regulatory reasons, have struggled and failed to appear as competent players in the global bio-economy of emerging technologies in the biosciences field. This investigation takes Mexico as a country case study to map out the factors hampering the development of the governance of emergent biomedical biotechnologies in this context, particularly that applied to stem cell science. This research aims to contextualise and portray prevailing ethical, legal, political and religious concerns regarding stem cell research in this context. Exploring the debates in these arenas, it seeks to elucidate the perceptions of key stakeholders and to appraise critically the divergences and convergences among the actors who currently shape the debate and who may have significant influence on the creation of any legislative framework in the area. It explores whether it is feasible to draw on the approach taken to stem cell science and tissue regulation in the United Kingdom, in order to illuminate the way forward for governing stem cell research and its clinical applications in Mexico. It also aims to evaluate the risks posed by the persistent lack of regulation in this scientific field, since Mexico appears to be an ideal destination for stem cell tourism among Latin American countries. Drawing on empirical data gathered from prominent Mexican stakeholders in the stem cell issue, this research elucidates the key themes influencing the debate which need to be addressed in detail in order to prepare the ground for the effective governance of stem cell science and its clinical applications. By detailing the emergent themes and providing reflexive explanations of the elements influencing the views of all the actors in this arena, this thesis aims to provide ethical, empirical and normative proposals to be translated by policymakers into purposive regulation of biomedical innovations. Thus, it delineates two main features of the debate over stem cell science regulation in Mexico and shows the urgent need to create a legal framework to deal with problematic situations provoked by the legal vacuum in this area: a) the legal inertia preponderant in the Federal Congress, which is mainly caused by the constant lobbying of politicians by the Roman Catholic hierarchy to endorse prohibitive policies in sensitive areas, such as sexual matters, reproduction and stem cell science; b) the increasing phenomenon of stem cell tourism in the country, requiring the adoption of ethical and legal measures to avoid potential physical and financial harm to desperate patients who seek stem cell treatments.In conclusion, I argue that it is plausible to advance a permissive model of governance for the area of stem cell science. This thesis is supported by the evidence gathered from stakeholders’ opinions, added to the data emanating from the analysis of the country case study. As a result, it is possible to propose as an initial strategy the adoption of significant regulatory features of the paradigmatic system of governance which applies in the United Kingdom. The law is up to date as of 19 June 2012.

The cancer stem cell hypothesis has recently re-emerged as a compelling paradigm for the development and progression of neoplastic disease. The hypothesis proposes that a specific subset of “cancer stem cells” (CSC), believed to share many features with normal stem cells, is exclusively responsible for maintaining tumour growth and driving progression. If the CSC hypothesis applies, it may require re-evaluation of the clinical approach to neoplasia. Spontaneous cancer in the domestic dog represents a significant welfare problem, with dogs developing many tumours strongly reminiscent of those affecting humans. This study sought to investigate whether cells with characteristics of CSC are identifiable in canine cancer. Assays to identify, isolate and characterise CSC were adapted to the canine system, and cancer cell lines and spontaneous tumours of diverse origin evaluated for the presence of candidate populations. Whilst analysis of surface expression patterns did not identify specific subpopulations within canine cancer cell lines, these were detectable in cells derived directly from primary tumours. Assays for stem cellassociated drug resistance mechanisms could also be used to identify subsets of putative canine CSC. Formation of “tumourspheres” by canine cancer cell lines was found to be highly density-dependent, so a potentially unreliable method of isolating CSC. Expression of the cell surface glycoprotein CD44 was associated with cellular proliferation status, although it may not represent a stable canine CSC marker. The NFκB survival pathway, associated with apoptosis resistance of some putative CSC, was constitutively active in canine cancer cell lines; suppression using specific inhibitors could reduce cell viability, indicating that this may represent a rational therapeutic target. Overall, these studies demonstrated that CSC assays may be adapted to the canine model system, although they require rigorous interrogation to distinguish apparent CSC attributes from basic biological properties. Cell lines have provided a stable background upon which to optimise assays, but appear less likely to demonstrate discrete CSC subpopulations. Putative CSC subsets may be more readily identifiable within heterogeneous primary tumour cells. The application of some of these adapted assays within a clinical setting may enable further characterisation of individual patients’ tumours, and inform therapeutic regimes for improved treatment outcomes.

The liver is the largest solid organ in the body and is frequently the site of injury. During disease, liver injury is usually compensated for by exceptionally efficient regeneration which occurs both from differentiated epithelia and also from an undifferentiated cell population with stem cell like qualities known as hepatic progenitor cells (HPCs). HPCs are particularly active during massive or chronic liver injury and therefore are an attractive target for much needed novel therapies to enhance regeneration in patients for whom the only current effective therapy is liver transplantation. Stem cells in other organs systems are believed to reside in a specialised microenvironment or niche which supports their maintenance and function. To investigate the hypothesis that HPCs are supported by a functional niche and are capable of regenerating hepatocytes, we commenced by establishing a number of murine in vivo models. Having shown a stereotypical niche, consisting of macrophages, myofibroblasts and laminin exists in both animal models and human disease, we investigated the active recruitment of extrahepatic cells into this niche and showed that macrophages are actively recruited from the bone marrow during liver injury. Macrophages were shown to influence HPC behaviour during injury. Furthermore using macrophages as a cellular therapy, induced HPC activation with corresponding changes to liver structure and function. Investigation of signalling pathways revealed and confirmed a TWEAK dependent activation of HPCs following macrophage transfer. Having demonstrated the potential for macrophage therapy via HPC activation, we aimed to study the ability of HPCs to regenerate the hepatic parenchyma. To do so we developed and characterised a novel model of hepatocellular injury and HPC activation. Using the genetic labeling of hepatocytes in this model we were able to show rapid and large scale repopulation of hepatocytes from a precursor source with HPCs being the critical precursor source of hepatocellular regeneration. In addition this process is again dependent on TWEAK signalling, without which HPC mediated regeneration fails resulting in mortality. Therefore HPCs are an attractive biological target for regenerative medicine, and both TWEAK signalling and autologous macrophage infusion offer genuine potential to manipulate these cells as future therapies.

The homeobox gene Pitx3 has been implicated as a key regulator for lens development because homozygous mutant aphakia mice, which are hypomorph for Pitx3, fail to develop lenses. One aim of my thesis is to investigate the underlying cellular and molecular mechanism of Pitx3 mediated lens defect by studying knockout mice lacking Pitx3. Chimeric embryos, generated by aggregating the wild type embryos with Pitx3 heterozygous or Pitx3 homozygous mutant ES cells, have been used to analyse lens development. Pitx3 null cells failed to colonise the lens epithelium in Pitx3 null wild type chimeric lens, suggesting that Pitx3 is cell-autonomously required for lens epithelial cells. Further study of Pitx3 null mice revealed an earlier downregulation of the lens epithelial markers PDGFR-alpha and E-cadherin in E11.5 lens epithelium, suggesting the loss of lens epithelial identity in Pitx3 deficient mice. Furthermore, cell cycle inhibitors p27KIP1 and p57KIP2 were ectopically expressed throughout the morphologically normal Pitx3 mutant lens vesicle, suggesting that inactivation of Pitx3 leads to cell cycle exit of epithelial lens cells. In addition, precocious activation of the fibre cell-specific proteins beta- and gamma-crystallins was observed in Pitx3 null lens. Beta-crystallin expression could be observed as early as E10.5 throughout the entire Pitx3 null lens vesicle and gamma-crystallin was detected in the malformed Pitx3 deficient lens at E11.5. RNA in situ hybridisation study revealed that the expression of the transcription factor Foxe3 was lost in Pitx3 null lens at E10.5, suggesting that Pitx3 maintains the lens epithelial cells partly via the regulation of transcription factor Foxe3 during lens development. Accordingly, this study provides the cellular and molecular basis for the lens defect observed in Pitx3 null and Pitx3 hypomorph aphakia mice. Pitx3 is a key transcription factor for the maintenance of lens epithelium and its absence leads to premature activation of fibre cell differentiation programme of lens epithelial cells. In the other part of my PhD, I have further developed the Pitx3-GFP knockin ES cell system with a goal to use this tool for the identification of determinants of midbrain dopaminergic (mDA) neurons, the type of cells lost in Parkinson’s disease (PD) patients. Experimental cell therapy and clinical trials have shown that foetal midbrain tissues, but not tissues from other DA neuron containing regions, can functionally restore the lost mDA neurons when transplanted in Parkinson’s disease patients. Therefore, it is essential to coax mDA properties on stem cell-derived neurons when considering therapeutic development. Within the central nervous system, Pitx3 is expressed exclusively in mDA neurons. Using a Pitx3-GFP knockin mouse line previously generated in the laboratory I have derived heterozygous and homozygous Pitx3-GFP ES cells from mouse blastocysts. In keeping with previous findings in our laboratory, the heterozygous Pitx3-GFP (Pitx3GFP/+) ES cell-derived GFP positive cells of neuronal morphology can be detected after in vitro differentiation using the PA6 coculture system. Furthermore, I have shown that these cells express tyrosine hydroxylase and midbrain markers Engrailed-1 and Nurr-1, demonstrating their midbrain characteristics. I have also generated supertransfectable Pitx3GFP/+ ES cells to offer a rapid and efficient way to express a transgene episomally. The Cre-mediated inducible system of Pitx3-GFP reporter ES cells has also been developed in our laboratory and I have shown that they have high induction efficiency thus allows transgene activation in a temporally controlled manner. The Pitx3 null ES cells showed impaired potential to differentiate into mDA neurons thus they may be used to evaluate candidate Pitx3 downstream target by gain-of-function test. In summary, I have developed a Pitx3-GFP reporter ES cell system to identify mDA regulators functionally by in vitro differentiation.

Neural tissues have long been difficult systems to study. They contain a large variety of cell types organized into complex three-dimensional structures. This complexity has made it challenging to model these systems in vitro using classical tissue culture models. Recent advances have made it possible to take advantage of stem cells’ innate ability to differentiate into a variety of neural cell types to study the developing brain and spinal cord in ways not previously possible. In this thesis I present how I have used these new methods in stem cell biology to further our understanding of neurodevelopment and disease, and my work on further improving these new model systems.First, I used single cell transcriptomics to analyze the earliest stages of mouse dorsal interneuron development in a newly created in vitro differentiation protocol. By using this model system I was able to enrich our dataset for populations of interest, and use pseudotemporal analysis to find genes that may be responsible for helping to specify the different types of dIs present in the mouse spinal cord. I found 9 genes that we were able to verify using in situ hybridization in sections of developing mouse spinal cord. Next, I transitioned from two-dimensional models of spinal cord development into three-dimensional models of brain development. Recent studies in the brain organoid field have highlighted the importance of improving oxygen and nutrient delivery to the developing organoids. We tested both media flow and hyperoxygenation’s effects on brain organoid development, and found that while both have effects, the effect of hyperoxygenation is much larger. This presents a simple method of improving cell stress in current model systems. Lastly, I used classic genetic overexpression methods to try to create a better model of Alzheimer’s Disease using brain organoids. Mouse models and two-dimensional culture models have continually failed to provide effective treatments that translate into human clinical trials, hinting that these models are lacking some key component. By overexpressing mutant tau in brain organoids, we were able to replicate some classical AD pathology. Together these experiments represent a variety of approaches to addressing questions in current neurobiology.

Abstract: Intestinal epithelial damage and homeostatic cell shedding sends evolutionarily conserved growth signals to activate division programs in stem cells, which renews the epithelium to maintain barrier integrity. In addition to conserved growth and stress signaling, microbes and innate immune pathways regulate stem cell function during homeostasis and disease. For instance, the microbiome promotes stem cell proliferation and differentiation while overactive innate immune signaling causes stem cell hyperproliferation and exacerbates tumorigenesis. It is clear bacteria and immune pathways are important regulators of stem cell function, however the complexity of the microbiome has made it difficult to elucidate the specific effects individual bacterial species have on stem cells. In addition, the intestine is a heterologous tissue composed of multiple different specialized cell types, including intestinal stem cells, absorptive enterocytes and secretory enteroendocrine cells. Given the heterogeneity of cell types in the intestine and the limitations of vertebrate genetics, cell-type specific contributions of immune pathway activation on intestinal growth have been hard to determine, especially the impact of immune activity in stem cells. To determine how individual bacterial species and immune pathways impact stem cell function I used Drosophila melanogaster as a model system. Drosophila provides a number of advantages for this work. For example, Drosophila can be easily reared without a microbiome and associated with single bacterial species to determine their effects. In addition, Drosophila genetics allow for the perturbation of immune signaling in distinct cell types of the intestine, including stem cells. Using Drosophila, I identified the commensal bacteria Lactobacillus brevis as a potent stimulator of stem cell divisions and tumorigenesis. L. brevis altered the expression and intracellular localization of integrins in stem cells, leading to symmetric stem cell expansion. Next, I asked what the consequences of immune signaling in stem cells are by activating or inhibiting the immune deficiency (IMD) pathway. Activation of IMD in progenitor cells resulted in intestinal hyperplasia and exacerbation of tumorigenesis. Furthermore, inhibition of IMD reduced homeostatic proliferation and disrupted differentiation. Finally, I asked if the NF-kB family transcription factor Relish acts in stem cells to modulate damage response. Damage of the intestine results in elevated stem cell divisions to repair the injury. However, stem cell specific Relish depletion caused stem cells and their progeny to undergo apoptosis, rendering intestines incapable of effective epithelial repair, leading to host lethality. Thus, bacterial species can have profound effects on stem cell physiology and that immune pathways act directly in stem cells to modify proliferation, tumorigenesis, differentiation and epithelial repair responses.

Stem cells are vital for maintaining and regenerating tissues, and they are influenced by both internal factors and external signals from their niche. However, the molecular signals involved in the communication between stem cells and their niche, particularly in the hair follicle stem cell (HFSC) field, are not well understood. Recent findings have provided more insights into this crosstalk, and this dissertation focuses on the signaling between HFSCs and their vascular niche specifically.The first part of the study characterized the changes in the vascular niche during different stages of the hair cycle under homeostasis. The skin vasculature was found to form a horizontal plexus under the hair germ (HPuHG) and then disperse upon anagen onset. To investigate the two-way signaling between HFSCs and their vascular niche, we induced (1) Runx1 knockout in the epithelium using K14-CreERT2 driver (Runx1 KO) and (2) Alk1 knockout in the endothelium using Cdh5-CreERT2 driver (pan-endothelial Alk1EndoKO), both in adult mice to investigate changes in the other compartment. Both knockout mice exhibited delayed HFSC activation and accumulation of the HPuHG. Further investigation revealed that Alk1 represses BMP4 expression in the endothelial cells, which is supported by both BMP4-LacZ reporter allele and genomic evidence from scRNA-seq data of sorted endothelial cells and bulk RNA-seq data of sorted HFSCs in Alk1EndoKO mice. The rescue experiment using Alk1;BMP4dKO mice showed a partial rescue of the HFSC activation delay seen in Alk1EndoKO mice. The study also revealed that blood vessels, but not lymphatics, can inhibit HFSC activation through signaling by genetically perturbing the Alk1-BMP4 pathway in all endothelial cells or solely in lymphatic endothelial cells. Overall, these results suggested that blood vessels have a more significant impact and function as signaling niches for adult HFSCs, expanding the functional repertoire of endothelial cells in the skin. In addition to the stem cell-niche communication, this work also identified a novel K19+/VE-Cadherin+ perineurial population, revealing the heterogeneity of the VE-Cadherin+ lineage.

Hematopoietic stem cells (HSCs) emerge from a population of specialized endothelial cells in the aorta during embryogenesis. These cells then migrate to a temporary niche where they reside and proliferate for the rest of fetal development. This niche supplies growth factors and cytokines to the newly formed HSCs and consists largely of sinusoidal vasculature, stromal cells, and primitive immune cells. While it is generally understood that HSCs proliferate in the fetal niche, precise analysis of changes to individual HSCs during this time has proven difficult due to the inaccessibility of the tissue in mammals and low cell numbers. Here, I leveraged the advantages of zebrafish embryos to address these problems and analyze changes to HSCs induced by the early niche. I developed new transgenic tools to enable magnetic enrichment of rare embryonic populations and applied this to study the transcriptional profile of HSCs over the first days of development. I also performed high-resolution time-lapse imaging of HSCs as they colonized the niche. I discovered intimate and specific cell-cell interactions between primitive macrophages and HSCs. Macrophage interactions frequently led to either removal of cytoplasmic material and stem cell division, or complete engulfment and stem cell death. Macrophage interactions were stimulated by the presentation of Calreticulin on the surface of HSCs. Surface Calreticulin was induced as a result of cellular stress and reactive oxygen species accumulation in emerging HSCs. Stem cells with high stress displayed higher levels of surface Calreticulin and were fully engulfed by macrophages. Stem cells with low-to-moderate stress levels displayed moderate levels of surface Calreticulin and were stimulated to divide by macrophage-produced IL1β. Using cellular barcoding, I found that Calreticulin knock-down or embryonic macrophage depletion reduced the number of stem cell clones that established adult hematopoiesis. My work indicates that the fetal niche serves to both supply proliferative factors for the emerging HSC pool, but also monitor stem cell quality and shape the clonal architecture of the stem cell population through interactions with primitive macrophages. This mechanism could be manipulated to therapeutically target specific cells in other high-stress environments, as in hematologic disease or tissue regeneration.

This doctoral thesis explores the remarkable domain of mechanobiology, transcriptional regulation, and its relevance in regenerative medicine. Cells are known to be plastic, capable of transitioning between different states based on their microenvironment. Mechanical cues, in particular, have emerged as a potent trigger for cell-state transitions, such as reprogramming and differentiation, offering exciting prospects for regenerative medicine. However, the molecular mechanisms underlying these mechanically induced transitions remained poorly understood. The first chapter discusses recent work on exploring the role of mechanical environment and nucleus mechanisms in transcriptional regulation in various cell state transitions. The second chapter unveils the power of laterally confined growth of fibroblasts in inducing dedifferentiation programs. The molecular mechanisms underlying mechanically induced cell-state transitions are illuminated, with a focus on the critical role of the somatic transcription factor, Lef1. Lef1 has been implicated in various cellular contexts, but its central role in mechanically induced fibroblast dedifferentiation emerges as a key finding. Network optimization methods applied to time-lapse RNA-seq data identify Lef1-dependent signaling as potential regulators of these transitions, shedding light on Lef1's interaction with downstream reprogramming factors. Moreover, it identifies Smad4 and Atf2 as potential critical activators of Lef1, establishing an important mechanotransduction pathway for fibroblast dedifferentiation. Building on this foundation, the thesis dives into the rejuvenation of fibroblasts - a key focus for functional tissue regeneration. Traditional methods of cellular rejuvenation can be limited, often necessitating complete reprogramming and carrying risks like genomic mutations and low efficiency. However, the mechanically induced partially reprogrammed spheroids offer an innovative solution. When embedded in 3D collagen matrices with optimized stiffness, these spheroids regain fibroblastic properties and transform into 3D connective tissue networks. These redifferentiated fibroblasts exhibit reduced DNA damage, enhanced cytoskeletal gene expression, and acto-myosin contractility. Besides, increased deposition of matrix protein and enhanced collagen remodeling indicate that these redifferentiated fibroblasts are rejuvenation. This work highlights efficient fibroblast rejuvenation through mechanical reprogramming, promising novel approaches in regenerative medicine. We next investigate mechanical reprogramming and redifferentiation with the goal of presenting a cellular rejuvenation approach for aged dermal fibroblasts. Aging is characterized by cellular functional decline and epigenetic changes, necessitating innovative rejuvenation strategies. Mechanically rejuvenated aged fibroblasts reset their global transcription profile, upregulating genes related to cell proliferation and extracellular matrix secretion. Innovative Hi-C analysis reveals genes associated with aging and rejuvenation within reorganized interchromatin contact regions. Imaging experiments unveil chromatin compaction changes, and novel measurements based on Lamina-associated domains (LAD) interactions offer insights into 3D chromatin organization. The findings introduce a multi-omics analysis approach and contribute to the understanding of chromatin-mediated cellular rejuvenation, with potential therapeutic implications. Finally, we consider the context of tissue regeneration and wound healing, where cell-based therapies play a pivotal role. Traditional autologous transplantation is hampered by cellular senescence and reduced remodeling capabilities. The thesis presents an alternative approach, implanting partially reprogrammed aged human dermal fibroblasts into in vitro aged skin models for tissue regeneration and wound healing. The implanted cells exhibit enhanced extracellular matrix protein expression and synthesis, leading to improved tissue regeneration at wound sites. Transcriptome analysis and chromatin biomarkers unveil the upregulation of tissue regeneration and wound healing pathways, offering a novel, non-genetic approach for cell-based therapies in regenerative medicine.

Due to the mechanosensitive nature of stem cells, intense research has also focused on the role of the cell microenvironment in directing cell fate decisions and stemness acquisition. It has been demonstrated that modulation of biophysical cues such as lateral confinement or substrate stiffness can enhance induced pluripotent stem cell (iPSC) and cancer stem cell (CSC) generation. While biophysical studies commonly involve modulating cell-cell and/or cell-ECM adhesions, a systematic study on the effect of perturbing cell adhesion during reprogramming has not been performed. Therefore, we propose to study the effect of a collection of cell-cell and cell-ECM associated genes, known as the Adhesome, on stemness acquisition of reprogramming cells, in the context of iPSC and CSC reprogramming. Here, we report on a custom RNAi screen targeting 103 adhesome genes which demonstrated that adhesome expression is largely refractory to somatic reprogramming and revealed two novel top hits not previously associated with stemness acquisition: SHROOM3 and CSRP1. scRNA-seq was performed on reprogramming fibroblasts and A549 cells with SHROOM3 and CSRP1 knockdowns, respectively. Adhesome expression knockdown in both reprogramming models decreased PCP and EMT signatures, increased stemness acquisition, and, as revealed by CellChat inferred communication patterns, utilized similar cell-environment signaling during stemness acquisition. Furthermore, trajectory inference of the adhesome gene knockdown in both reprogramming models revealed novel pathways to stem-like states and cell fate transitions. Together, our findings support the adhesome as a novel target during somatic and oncogenic dedifferentiation for the development of more effective stem cell therapies and cancer treatments.