Late-onset retinal macular degeneration (L-ORMD) is a fully penetrant autosomal dominant macular degeneration resulting from a Ser163Arg substitution in the gene encoding the protein C1QTNF5. Clinically L-ORMD results in dark adaptation delay in the fifth decade, central visual loss in the sixth decade and further progressive visual field loss in successive decades of life. Pathologically the disease results in thick sub-retinal deposits, which have a similar composition to drusen seen in AMD, retinal pigment epithelial (RPE) loss, and neuro-retinal atrophy. The function of C1QTNF5 is incompletely understood however within the eye it is expressed most strongly by the RPE cells. An in vitro model for L-ORMD was developed using human induced pluripotent stem cells (hiPSCs) derived from patients and with stem cells from patient’s unaffected siblings used as controls. The hiPSCs were differentiated to RPE (hiPSC-RPE). L-ORMD hiPSC-RPE shared baseline characteristics with sibling control hiPSC-RPE. In order to model in vivo conditions hiPSC-RPE were grown on permeable supports in human serum enriched media. Case hiPSC-RPE cell lines were found to activate the complement pathway resulting in increased deposition of the terminal complement complex (TCC) C5b-9 when compared to control hiPSC-RPE. Using depleted serum, deposition was not affected by depletion of classical and lectin pathway components but was reduced by depletion of alternative complement pathway components. Depletion of complement components C3 and C5 abolished TCC deposition. The addition of a monoclonal antibody against C5 also reduced TCC deposition. The role of complement dysregulation in L-ORMD pathogenesis was confirmed by immunostaining of L-ORMD and age-matched control human donor retinal sections. L-ORMD retinal sections displayed increased C3d and C5b-9 deposition. Using mutant and wild type-protein generated from a bacterial expression system it was found that the mutant protein was less stable than the wild-type. In addition the wild type protein formed multimers whilst the mutant was mainly monomeric. A surface plasmon resonance (SPR) study showed an increased affinity of wild-type C1QTNF5, especially in multimeric form for complement factor H (CFH), a key regulator of the alternative complement pathway when compared to mutant protein. Taken together these studies implicate dysfunction of the alternative complement pathway in L-ORMD disease mechanism and have suggested a role for C1Q TNF5 in the extracellular matrix. The studies also show that L-ORMD and AMD share a pathogenic and clinical similarities.

In adult mammals, the gastrointestinal (GI) epithelium exhibits the highest turnover rate among the endodermal tissues. The harsh luminal environment of the GI tract necessitates replenishment of epithelial cells to maintain organ structure and function during routine turnover and injury repair. This delicate balance between gain and loss of cells is called tissue homeostasis, and multipotent tissue specific adult stem cells serve as the continuous source of self-renewal. Due to their important contribution to homeostatic maintenance the proliferative capacity of the stem cells needs to be tightly controlled, as an imbalance can result in diverse pathologies such as cancer or insufficient injury repair. Despite the crucial role for regulatory processes the molecular mechanisms and the genes governing these processes remain poorly understood. Rnf43 and its paralogue Znrf3 (RZ) act as tumour suppressors in the intestine, but their role in the gastric epithelium has not been previously investigated. Using a novel unpublished stomach specific CreERT2 expressing mouse line I found that simultaneous knockout of RZ (RZ DKO) result in gastric hyperplasia of the corpus epithelium. Gastric RZ DKO organoids show independence from the essential growth factor Rspondin-1 but require exogenous Wnt. A similar exogenous Wnt dependence was identified in a human gastric cancer cell line harbouring homozygous Rnf43 inactivating mutations. Thus, Wnt secretion inhibition might provide a new treatment paradigm for a subset of patients carrying Rnf43 mutations. The prominent role of the E3s Rnf43 and Znrf3 in the intestinal and gastric epithelial led to the question of whether other E3s either closely related to RZ or specifically expressed in stem or niche cells could play a role in homeostatic regulation, specifically in the small intestine. Using a retroviral overexpression screen I identified Rnf24 and Rnf122, two E3s that rendered intestinal organoids insensitive to withdrawal of the BMP inhibitor Noggin. Moreover, potential substrate candidates located at the cell surface membrane were identified and the generation of in vivo models initiated to provide a basis for further studies investigating the role of these E3s. In trying to address the function of the abovementioned genes using in vitro functional genetics I identified gaps in the current technology for organoid genetic engineering. I therefore developed two gene editing methods; a gRNA concatemer system allowing simultaneous knockout of multiple genes and CRISPR-FLIP enabling generation of conditional gene knockouts In summary, this thesis describes the first stomach specific knockout of Rnf43 and Znrf3 in the gastric epithelium, showing that it results in gastric hyperplasia located to the corpus epithelium. The dependence of the Rnf43 and Znrf3 knockout epithelium on exogenous Wnt signalling provides a potential treatment strategy for a subset of patients harbouring Rnf43 mutations. Next, it identifies Rnf24 and Rnf122 as E3 ubiquitin ligases involved in intestinal stem cell regulation and provide preliminary data and a basis for future studies. Finally, it describes the establishment of two advanced genetic engineering approaches which can be applied to various in vitro culture systems such as 3D organoids, mouse embryonic stem cells and conventional cell lines. Collectively this work and the developed methods will contribute to our understanding of the mechanisms regulating adult stem cell homeostasis.

This thesis presents the development of microfluidic devices designed to facilitate research into mouse embryonic stem cells (ESCs). ESCs are a well-studied cell, largely due to their pluripotent nature, meaning they are able to differentiate into all cell types of the body and may self-renew indefinitely in appropriate culture conditions. ESCs, along with many other lines of biological enquiry, are increasingly studied with the use of micro uidic technology which enables fine tuning of physical and chemical environments unachievable on the macro scale. Two varieties of microfluidic technology are presented in this thesis, one for high- resolution mechanical phenotyping of ESCs and the second as a novel in-chip culturing platform to study cellular transitions. Chapter 1 presents a broad introduction to ESCs and biological enquiry with microfluidics, aimed to underpin the following Chapters. Chapters 2 and 3 present self-contained projects, thus each include a motivation and introduction section more specific than that presented in Chapter 1. These Chapters also contain their own methods, results and conclusion sections. Finally, Chapter 4 presents a summary of the work performed along with an outlook of upcoming investigations. In Chapter 2, I present a microfluidic device developed and utilised in collaboration with Christophe Verstreken (Department of Physics, University of Cambridge), which has been used to apply a mechanical stress to live cells enabling measurement of their nuclear deformability. The device facilitates detection of both nucleus and cytoplasm which can then be analysed with a custom-written MATLAB code. Quantitative measurements of nuclear sizes and strains of ESCs indicated a negative Poisson ratio for nuclei of cells cultured in specific medium conditions. Furthermore, we demonstrate that the device can be used to physically phenotype at high-throughput by detecting changes in the nuclear response after treatment with actin depolymerising and chromatin decondensing agents. Finally, we show the device can be used for biologically relevant high-resolution confocal imaging of cells under compression. The work from this chapter is presented in Hodgson et al. [1]. In Chapter 3, I present a novel microfluidic platform developed in collaboration with Prof. Austin Smith and Dr Carla Mulas (Centre for Stem Cell Research, Cambridge). The developed platform enables individual ESCs to be cultured under continued observation as they exit their pluripotent stem cell state. Each cell within the device may be extracted from the chip at any time for further investigation without disturbing other cells. Assessing the transition from the stem cell state in individual cells is paramount if we are to understand the mechanisms of pluripotency.

Patients with Acute Myeloid Leukaemia (AML) present with the signs and symptoms of bone marrow failure. This finding spans the genetic and phenotypic diversity of the disease. The mechanism which underlies it is poorly understood. This thesis explores the effect of AML on the normal haematopoietic stem cell (HSC) population, using primary human diagnostic bone marrow samples. Previous work from our group suggested that AML induces a state of quiescence in HSCs, producing a differentiation block responsible for the observed cytopenias1. Reversal of this process might offer an alternative to the current treatment of patients with palliative transfusions. I have developed a flow cytometry-based technique to differentiate normal HSCs from leukaemia cells, selecting cells with the CD34+38-ALDHhighCLL1- expression signature. Validation of this technique by assessment of sorted cells by FISH and PCR, suggests it is successful in 73% of AML samples. In a further 25% of samples, it selects for a population significantly enriched for normal HSCs. We used this panel to investigate the concentration of HSCs at AML diagnosis, compared to controls. We show that there is no significant difference between HSC concentration at AML diagnosis (n=38, median [HSC] 2.5 cells/μl) and controls (n=24, median [HSC] 2.4 cells/μl). HSC concentration was not significantly affected by AML karyotype, patient age or gender. However, those patients presenting with a low HSC concentration at diagnosis (< 0.1 HSC/μl) were found to have a significantly worse outcome both in terms of overall and relapse-free survival, an effect apparently independent of age, gender and underlying karyotype. HSC concentration at diagnosis with AML may therefore represent a new independent prognostic marker. We then studied CD33 expression patterns on HSCs within Core Binding Factor mutated AML (n=37) at diagnosis, and found its expression to be significantly lower than on HSCs within controls (n=9) (17% versus 58%, p=0.005). CD33 expression on HSCs from AML samples rose significantly from diagnosis to remission (n=16) (17% to 58%, p=0.0001). This mirrors previous findings from our group using CD34low AML samples, and is, we believe, the first time that the antigenic signature of normal HSCs has been shown to be modified. 6 by the presence of AML. However, an in vitro assay to test the significance of these changes in terms of the cytotoxicity of GO towards normal HSCs did not demonstrate a significant difference between HSC subgroups. Finally, we attempted to investigate the mechanism by which AML might induce HSC quiescence by studying the comparative transcriptomes of HSCs from CD34low AML (n=6) and controls (n=6) by RNA-Seq, using direct cell to cDNA synthesis, followed by amplification. A first attempt resulted in poor quality data, with a significant proportion of reads mapping to non-coding DNA regions. A repeat approach, using utilising immediate RNA extraction post sorting resulted in significantly better quality data Bioinformatics analysis revealed differential expression of 6 genes between the 2 datasets (GNPDA1, ADGRG3, MIAT, WDR31, RP11-244H3.1 and RXFP1). GO enrichment studies using David highlighted a number of pathways including the TNF signalling pathway (p=0.003; after Benjamini-Hochberg correction p=0.51). Validation of these findings by independent qPCR, and functional exploration of enriched signalling pathways remains outstanding.

皮膚是人體最大的器官，具有多種功能，其中最重要的功能之一就是作為身體內部和外界環境之間的的保護屏障。完整地修復這一保護屏障是創傷癒合和組織再生領域的一個重要內容。本論文探討了人類臍帶被覆上皮細胞 (cord lining epithelial cells, CLECs)作為一種幹細胞來源，可用于表皮重建的潛能. / 本論文的第二章對CLECs的體外分離和增殖進行了詳細地描述。這一類細胞具有較長的染色體端粒，較高的增殖潛能和傳代能力。同時，它們表達上皮幹細胞和多能性幹細胞的標誌性表面抗原。它們還具有多種分化潛能，包括成脂、成骨和成軟骨。然而當皮下異種移植後，它們並不會形成畸胎瘤。 / 本論文的第三章對CLECs的免疫特性進行了評估。結果顯示CLECs不但具有低免疫原性，還具有免疫調節功能。它們表達典型性的一型主要組織相容性複合體(MHC class I)，即人白細胞ABC抗原(HLA-ABC)，但不表達典型性的二型主要組織相容性複合體(MHC class II)，即人白細胞DR抗原(HLA-DR)。它們同時還表達非典型性的MHC class I, 包括人白細胞G抗原和人白細胞E 抗原(HLA-G和HLA-E), 但不表達共激分子(CD40, CD80和CD86)。此外，體外檢測還發現它們表達適度的促炎/抗炎細胞因子和大量的生長因子. / 本論文的第四章對CLECs在表皮重建應用中的潛能進行了考察。結果顯示無論在體外器官培養還是異種移植動物模型中，CLECs都能形成分層的上皮結構，與用表皮細胞構建的分層上皮結構相類似。而且在CLECs構建的皮膚替代物中證實了有表皮分化標誌性抗原的表達。 / 結論：本論文證明了CLECs具有幹細胞樣特性但無致瘤性，具有低免疫原性和表皮分化的可塑性。研究結果支持CLECs在創傷癒合和皮膚再生領域的臨床應用可行性. / The skin is the largest organ in the body and has multiple functions. One of the most important functions is to serve as a protective barrier between the internal and external environments of the body. Restoration of the integrity of this protective barrier is an essential aspect of wound healing and tissue regeneration. In this thesis, the potential of human umbilical cord lining epithelial cells (CLECs) as a source of stem cells with appropriate differentiation capacity for epidermal reconstitution has been explored. / The isolation and propagation of CLECs from human umbilical cord lining epithelium were described in Chapter II. The cells presented a long telomere length and had high proliferative potential and passaging capability. They were also shown to display both epithelial and pluripotent stem cell markers. They were capable of multipotent differentiation, including adipogenesis, osteogenesis and chondrogenesis. However, they didn’t form teratoma after subcutaneous xenotransplantation until 12 weeks. / The immune properties of CLECs in vitro were assessed in Chapter III. The cells were shown to have low immunogenicity but high immunosuppressive function. They expressed classical major histocompatibility complex (MHC) class I antigens (HLA-ABC), but not MHC class II antigen (HLA-DR). They also expressed non-classical MHC class I antigens (HLA-G and HLA-E), but lacked the expression of the co-stimulatory molecules (CD40, CD80 and CD86). Moreover, they expressed moderate pro/anti-inflammatory cytokines and multiple growth factors both in cell supernatants and cell lysates. / The potential of CLECs for epidermal reconstitution was investigated in Chapter IV. In both organotypic culture and xenotransplantation model, CLECs were capable of generating a stratified epithelial structure, which is similar to that constructed by using keratinocytes. Furthermore, the expression of epidermal differentiation markers was verified in CLEC-constructed skin substitutes. / In conclusion, the stem cell-like properties of CLECs have been demonstrated in the present study. In addition to the lack of tumorigenicity, CLECs also have low immunogenicity and significant plasticity in epidermal differentiation. The findings support the potential clinical application of CLECs in wound healing and skin regeneration. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cai, Yijun. / "October 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 114-129). / Abstract also in Chinese. / Abstrac --- p.i / Table of Contents --- p.v / Abbreviations --- p.vii / List of Figures --- p.viii / List of Tables --- p.x / Chapter Chapter I --- Introduction --- p.1 / Skin --- p.3 / Wound healing --- p.6 / Wound regeneration and repair --- p.6 / Recent history of wound treatment --- p.9 / Skin substitutes --- p.11 / Stem cells for wound treatment --- p.14 / Stem cells overview --- p.15 / Adult stem cells --- p.16 / Fetal stem cells --- p.18 / Amniotic membrane derived stem cells --- p.19 / Umbilical cord stem cells --- p.22 / Hypothesis and Specific aims --- p.24 / Chapter Chapter II --- The Isolation and Characterization of the Stem Cell-like Properties of Human Umbilical Cord Lining Epithelial Cells --- p.28 / Introduction --- p.28 / Materials and methods --- p.30 / Results --- p.47 / Discussion --- p.62 / Conclusion --- p.67 / Chapter Chapter III --- The assessment of the Immune Properties of Human Umbilical Cord Lining Epithelial Cells --- p.69 / Introduction --- p.69 / Materials and methods --- p.72 / Results --- p.75 / Discussion --- p.83 / Conclusion --- p.88 / Chapter Chapter IV --- The Investigation of the Potential of Human Umbilical Cord Lining Epithelial Cells for the Epidermal Reconstitution --- p.89 / Introduction --- p.89 / Materials and methods --- p.91 / Results --- p.94 / Discussion --- p.101 / Conclusion --- p.104 / Chapter Chapter V --- Summary and Future Plan --- p.105 / Summary --- p.105 / Future plan --- p.108 / Acknowledgements --- p.113 / References --- p.114 / Appendix --- p.130

Cancer is a highly complex disease that evolved in response to a wide range of biological and molecular changes that impact disease behaviour, treatment efficacy and clinical outcomes. Studying this diversity in human tumours is essential for gaining insights that will ultimately improve the survival rates of cancer patients. Cancer stem-like cells (CSCs) are believed to be responsible for invasive and metastatic features in tumours and can contribute to chemotherapy resistance and subsequent tumour relapses. There is an increasing need to identify the molecular mechanisms involved in tumour cells, particularly in CSCs. Cancer testis antigens (CTAs) are a subclass of germline proteins normally produced in immune-privileged sites, such as the testis, ovary and placenta of somatic tissues, and the presence of these antigens is increased in a variety of cancers. These characteristics make CTAs highly important immunotherapeutic targets, since they do not harness the immune response in the testes but encode immunogenic proteins that can induce a specific response in cancerous tissues. CTA genes are potentially very importance in clinical applications, including cancer diagnosis, vaccination and immunotherapy. This current study focused on the investigation of two CTAs, TDRD12 and LKAAEAR1, that may have an enhanced presence in cancer and the potential to be immunogenic. TDRD12 is linked to stemness features and enables the proliferation of germ line tumour cells. It appears to act as a possible transcriptional regulator for germline factors that are essential to cell cycle proliferation, germ cell maintenance and stem marker expression. TDRD12 may have the potential to drive oncogenesis and CSC targets. LKAAEAR1 was validated as a CTA at the protein level, showing its production was restricted to germ cells and the central nervous system from normal tissues and showed aberrant production in a wide range of tumours. This protein has been shown to be produced in germ cells undergoing spermatogenesis with strong nuclei staining, suggesting its potential role in this process. LKAAEAR1 potentially acts as a regulator for transposable elements, thereby increasing its contributions to cancer development. This study demonstrated that LKAAEAR1 could potentially be used as a cancer biomarker and therapeutic target.

Self-renewal mediates homeostasis across mammalian organ systems as the cellular components of mature tissues are continually replaced in the face of wear and tear, injury, infection, and malignancy. The hematopoietic and immune systems are crucial for organismal longevity and rely on the ability of progenitor cells to bifurcate in fate to produce mature terminally differentiated progeny while self-renewing to maintain more quiescent progenitors. Asymmetric cell division is associated with self-renewal of lymphocytes and hematopoietic progenitors, but the mechanisms underlying the cell biology of these processes remain incompletely understood. Here we show that metabolic signals in the form of differential anabolism and catabolism regulate asymmetric division and cell fate bifurcations. Key transcription factors, including TCF1 and IRF4 in lymphocytes and IRF8 in hematopoietic progenitors, occupy regulatory nodes where signals associated with metabolism and traditional cell fate determinants converge. Notably, anabolic PI3K/mTOR signaling was required for terminal differentiation of both lymphocytes and hematopoietic progenitors through the regulation of a constellation of nutrient uptake, mitochondrial turnover, reactive oxygen species production, and autophagy. Further, we found that antigen receptor signaling in lymphocytes organizes a cell-intrinsic polarity pathway of asymmetric intracellular membrane trafficking that is regulated by PI3K activity and associated with terminal differentiation. These results support a model wherein cell fate bifurcations are organized by metabolic signaling at the population and subcellular level to ensure self- renewal of progenitor and memory populations.

The decisions stem cells make impact both the development of adult vertebrates and systems within the body that require cellular replenishment to sustain life. Regardless whether a stem cell remains quiescent, divides, differentiates, or undergoes apoptosis—these processes are precisely controlled by internal gene regulatory networks that are instructed by external stimuli. The exact mechanisms governing stem cell fate are not completely understood. These studies explore new ways in which cell fate is mediated. Through a study of mitochondrial content in human embryonic stem cells (hESCs) and their differentiated progeny, we discovered differences in mitochondrial morphologies. Mitochondria began as elongated and networked structures in self-renewing conditions and changed their shape after differentiation. The addition of external growth factors that direct hESCs toward the definitive endoderm (DE) lineage promoted mitochondrial fragmentation, which was mediated by the mitochondrial fission machinery. Globular, punctate mitochondria were observed prior to the induction of the DE-specific transcriptional program. Differentiation of hESCs to other lineages did not result in any mitochondrial shape changes. Thus, mitochondrial fission in differentiating hESCs, an internal cellular process, is induced by DE-inducing external stimuli, an effect that was lineage specific. In a second study, we investigated the role of the splenic environment in the development of the blood system—during hematopoiesis. The spleen made a distinct contribution to hematopoiesis, a process predominantly attributed to the bone marrow. We discovered a previously unidentified population of cells, uniquely represented in the mouse spleen that could develop into erythrocytes, monocytes, granulocytes, and platelets. These multipotent progenitors of the spleen (MPPS) expressed higher levels of the transcription factor, NR4A1 compared to their bone marrow counterparts and relied on NR4A1 expression to direct their cell fate. The activation of NR4A1 in MPPS biased their production of monocytes and granulocytes in vitro whereas NR4A1-deficient MPPS over-produced erythroid lineage cells in vivo. Together, these data suggest the splenic niche supports distinct myeloid differentiation programs of multi-lineage progenitors cells. Both studies identify new mechanisms by which external stimuli regulate internal mechanisms of cell fate. These insights provide a better understanding of stem and progenitor cell differentiation that have the potential to impact cellular replacement therapies.

Adult stem cells are vital to animal biology, tasked with replenishing cells in a variety of tissue types. Historically, there have been two contrasting models of stem cell behavior, “single-cell asymmetry,” where each stem cell is generally long lived and is responsible for perpetual daughter (non-stem) cell production, and “population asymmetry,” where a group of stem cells maintain population numbers while producing non-stem cell daughters, but individual stem cells undergo stochastic competition leading to frequent loss or amplification of individual lineages. Our work examines Drosophila ovarian Follicle Stem Cells (FSCs), which are somatic adult stem cells organized as a population of 14-16 cells within each germarium. FSCs are responsible for the production of two distinct somatic daughter cell types at opposite borders of the stem cell population. The FSCs are arranged in three anteroposterior layers; posterior “layer 1” FSCs divide faster and directly produce Follicle Cells (FCs), while anterior “layer 2 and 3” FSCs divide more slowly and give rise to Escort Cells (ECs). We have examined how signaling pathways contribute individually to FSC behavior, using clonal analysis to manipulate individual FSC genotypes and subsequently determine how autonomous FSC properties and competition among FSCs is affected. Our data indicate that Janus Kinase/Signal Transducers and Activators of Transcription (JAK-STAT) and Wnt pathways are primarily responsible for regulating location, proliferation and differentiation in FSCs. The activities of Hedgehog (Hh), Hippo (Hpo), and Phosphoinositide 3-kinase (PI3K) pathways are also shown to be important for FSC competition.

Signals for each of the five tastes have previously been shown to be processed by distinct labeled lines from taste receptor cells (TRCs) on the tongue to the ganglion neurons that innervate them. Furthermore, different tastes have been shown to be represented by distinct neurons in the taste cortex. We recorded calcium activity using fiber photometry from genetically defined populations in the mouse rostral nucleus of the solitary tract (rNST), the first brain station receiving taste signals from the tongue. We found that Somatostatin- (Sst) expressing cells respond exclusively to bitter chemicals while Calretinin- (Calb2) expressing cells respond exclusively to sweet chemicals. Immunostaining and viral strategies demonstrated that Sst and Calb2 mark distinct neuronal populations in the rNST. We then showed that optogenetic activation of Sst and Calb2 cells elicits prototypical bitter and sweet behaviors, respectively and demonstrate that ablation of these cells strongly impairs aversion to bitter tastants and attraction to sweet tastants, respectively. These findings reveal how taste information is propagated into the brain.