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.

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Stem cell pluripotency and self-renewal are two important attributes of human embryonic stem cells which have led to enhanced interest in stem cell research. Understanding the mechanisms that underlie the regulation and maintenance of these properties is imperative to the clinical application of stem cells. Pluripotency and self-renewal are regulated by different genes, transcription factors and other co-factors such as FoxD3 and Klf4. Oct4, Nanog and Sox2 are central to the stem cell regulatory circuitry. They form interactions with co-factors to promote cell proliferation and inhibit differentiation by negatively regulating differentiation markers. However, there are other novel pluripotency associated factors yet to be studied. In this study, bioinformatics and functional analyses were employed to identify a potential pluripotency gene called YY1AP1 from our lab's pre-existing microarray data. YY1AP1, a transcription regulatory gene, showed consistent down-regulation with induced cell differentiation. It was further investigated. First, its co-localization with Oct4 in both hESCs and iPSCs was confirmed by immunofluorescence staining. Knockdown experiments were then performed on this gene to investigate effects of knocking it down on gene expression in hESCs. Knocked-down cells were characterized for markers of pluripotency and differentiation at the transcript level. Results showed a down-regulation of pluripotency genes with no specific promotion of any of the germ layer markers. Gene expression at the protein level in knocked down cells was then assessed for YY1AP1, and its binding partner YY1, and pluripotency markers. Results showed that proteins of YY1AP1, YY1, Oct4, Nanog and CTCF were down regulated while the tumour suppressor gene protein, p53, was up-regulated in YY1AP1 deficient stem cells. Protein to protein interaction studies showed that YY1AP1, YY1, Nanog and CTCF proteins directly interacted with each other. Differentiation of YY1AP1deficient cells into EBs led to an almost complete shutdown of all gene expression, an indication that the cells did not form 'real' EBs. Differentiation of YY1AP1 ablated cells did not support any lineage promotion either. These results suggest a potentially new role for YY1AP1 in proliferation and self-renewal of stem cells through its possible direct binding to CTCF or its indirect binding to CTCF in complex with YY1.

The aim of this thesis is to investigate different functionalization strategy of graphene nanomaterials for graphene-based different biotechnological applications such as graphene-directed stem cell growth and differentiation and graphene-based biosensors. Chemical functionalization of graphene is required in many biological applications; in this thesis we have focused on exploiting the carboxylic groups available on GO molecules and non-covalent functionalization of graphene. GO has been a promising material for stem cell culture due to high specific surface area, ease of functionalization, its ability to support cell proliferation and to not cause cytotoxicity when stem cells are cultured on its substrate. The impact of biochemical functionalization on stem cell differentiation was not widely researched, and many research groups worldwide have been focusing on GO and rGO surfaces only. The approach of this thesis is to fabricate and characterize different graphene-based substrates to investigate the impact of biochemical functionalization of GO in directing adipose stem cell differentiation and to influence the gene expression pathways of Schwann-like differentiated adipose stem cells. The fabrication of graphene based biosensors is still challenging as biological molecules need to be attached to graphene-based sensors to increase both the specificity and the selectivity of the biosensors. In this thesis, two different chemical functionalization approaches were considered. Firstly, the covalent immobilization of membrane proteins embedded on a lipid nanodisc structure on GO was achieved. Secondly, the feasibility of using dip-pen nanolithography as a tool to locally functionalize graphene arrays with phospholipids was demonstrated. Phospholipid interface layer can act as bioactive layer which can be used for the protein insertion of tail-anchoring recombinant proteins as a new route for a non-covalent biological functionalization of graphene array.

Rearrangements involving the mixed lineage leukaemia (MLL) gene are found in 5-10% of human leukaemias and are likely propagated by a deregulated self renewing pool of leukaemia stem cells (LSCs). Targeting of the LSC pool represents a key novel strategy for the treatment of AML. In recent years epigenetic dysfunction has been identified as a key driving factor in a range of solid tumours and haematological malignancies. Evidence for this includes identification of mutations in the genes coding for critical epigenetic modifiers, characterisation of localised regions of abnormal chromatin at oncogene or tumour suppressor genes and the efficacious use of epigenetic-targeted therapies already present in the clinic. The data submitted in this thesis identify the histone demethylase KDM1A as a critical regulator of LSC potential in MLL-AF9 acute myeloid leukaemia (AML). Of all the histone demethylases, we found that only Kdm1a expression correlated positively and significantly with LSC frequency in murine models of human MLL fusion AML. Genetic knockdown or Cre-mediated excision of Kdm1a resulted in loss of LSC potential, reduced expression of LSC maintenance transcriptional programs and induction of macrophage differentiation in MLL-AF9 cells. These effects were phenocopied by chemical inhibition of KDM1A using the monoamine oxidase inhibitor tranylcypromine (TCP), as well as novel TCP analogues which inhibit KDM1A with greater potency and selectivity. These results were seen in murine, human cell line and primary patient cells harbouring MLL rearrangements. Global transcriptome and epigenome analyses revealed a key role for KDM1A in maintaining the histone three lysine four (H3K4) methylation status at highly expressed MLL-AF9-bound genes. In vivo transplantation of Kdm1a knockdown MLL-AF9 cells conferred a significant survival advantage compared with control littermates. Similarly, TCP analogue treatment of mice transplanted with MLL-AF9 cells revealed a reduction in LSC potential of the donor-derived AML cells but little impact on normal recipient haematopoietic stem and progenitor cells (HSPCs). Critically the clonogenic and repopulating potential of normal HSPCS, of both murine and human origin, was spared following either knockdown or chemical inhibition of KDM1A. Taken together, the data presented establish KDM1A as a potential therapeutic target in MLL fusion leukaemia.

Targeted differentiation of human embryonic stem cells (hESCs) towards definitive endoderm (DE) is the first step in generating hepatic or pancreatic cell types with potential for clinical application. Characterisation and efficiency of DE differentiation is improving, however the specific effects of the different exogenous growth factors used, and the changing presence and activity of endogenous factors, are still not well understood. One such endogenous factor, the TGFβ ligand Nodal, is known to drive patterning and differentiation of the primitive streak and DE in the developing mouse embryo. The effect of Nodal signalling during hESC DE differentiation is unknown, and the common use of a related exogenous ligand Activin A may also serve to upregulate rather than simply mimic it. In order to explore this, Activin A differentiation of hESCs in defined culture conditions was analysed. The expression of characteristic mesendoderm and DE markers increased during Activin A treatment, which was significantly enhanced by the inclusion of exogenous Wnt3a. A maintained presence of the pluripotency factor Nanog was observed in most cells expressing markers of DE. The levels of Nodal and its co-receptor Cripto, which were raised during the early stage of Activin A treatment, were also marginally enhanced by Wnt3a, and evidence of Nodal endocytosis further suggested an active signalling presence. RNA interference (RNAi) of Nodal negatively affected both pluripotency maintenance during normal pluripotent culture, and the capacity to differentiate towards DE. Use of a Cripto blocking antibody also inhibited differentiation towards DE. The results strongly suggested the presence of Nodal signalling, as well as possible roles for Nanog, Wnt-related signalling, and Nodal signalling during Activin A-mediated DE differentiation. The results contribute to current understanding of how DE differentiation in hESCs is regulated. They also identify clear targets for further investigation, which would lead to improved characterisation and differentiation of DE from hESCs.

In mammalian development, endoderm formation occurs in two phases and the fate of these populations is different. In the blastocyst, inner cell mass (ICM) cells generate the primitive endoderm (PrE), which will give rise to the extra-embryonic parietal (PE) and visceral endoderm (VE). Hematopoietically expressed homeobox (Hhex) protein is initially expressed throughout the PrE and subsequently becomes restricted to the anterior visceral endoderm (AVE), one of two important early embryonic signalling centres in the mouse. During gastrulation a second wave of endoderm differentiation occurs, the definitive endoderm (DE), generating the foregut. Immediately following the induction of DE, regional identity is initially established in the anterior region with the expression of Hhex. One of the earliest specification events in this lineage is the specification of anterior fate by Hhex, this time in a second signalling centre, the anterior definitive endoderm (ADE). The ADE is both important for embryonic patterning, and as the precursor population for differentiating to the foregut and its derivatives the thyroid, liver and pancreas. The literature surrounding these early embryonic patterning events is covered in depth in chapter 1. Embryonic stem cells (ESCs) are normal cell lines derived from the mammalian blastocyst at the time that it is making PrE. A number of laboratories have generated protocols to make endoderm from ESCs and in my thesis I define approaches to distinguish between PrE and DE. I generated a new ESC reporter line utilising a gene normally expressed in both the PrE and later in hepatic endoderm; this reporter contains a GFP in the first exon of the Hnf4α locus. This was combined with a second fluorescent reporter containing DSRed in the Hhex locus. This cell line is described and characterised in chapter 3. As Hnf4α is initially expressed in PrE prior to Hhex, but in the DE following Hhex, I was able to use the temporal expression of this reporter to distinguish the induction of PrE from DE. As Activin and Wnt are known to induce endoderm from ESCs, I was then able to ask what sort of endoderm the combination of these two signals induced. In chapter 4 I found that normal ESCs would readily differentiate to iPrE in the presence of Activin and Wnt3a. While this has not been described previously, my analysis suggests that ESC protocols applying these cytokines directly to ESCs have produced PrE. Given that ESCs are derived from the blastocyst, the generation of iPrE from Wnt3a/Activin treatment fits with developmental paradigms. However, Act/Wnt3a is used routinely on Human ESCs (hESCs) and so I attempted to reconcile these observations. HESCs, while derived from the blastocyst, appear to progress developmentally in vitro, to a stage closer to the epiblast, immediately prior to gastrulation. I therefore assessed the effect of Activin and Wnt3a on mouse stem cell lines derived from the epiblast (Epiblast Stem Cells, EpiSCs), that are grown under similar conditions to hESCs. When Wnt3a/Act is applied to these cells I found that they made DE rather than PrE, which I describe in chapter 4. Taken together my observations suggest that Act/Wnt3a are general endoderm inducers that induce context specific differentiation in vitro. The cell type derived in response to this treatment depends on the developmental stage of the starting stem cell culture. During the course of this work, I also observed that PrE was growing under Activin/Wnt3a treatment. As a number of cell culture systems have been established that reflect PE, but not truly bipotent PrE, I investigated the conditions under which PrE can be expanded. In chapter 5 I characterize a new PrE culture system, in which bipotent extra-embryonic endoderm can be expanded indefinitely in culture. I also explore a bit more precisely the nature of the starting cells that initially become exposed to Activin/Wnt3a treatment. Previous work has extensively characterized the existence of a primed population of PrE in ESC culture and in chapter 6 I explore the existence of a primed DE population in EpiSC culture. Taken together, my thesis is the first demonstration that Activin/Wnt3a can induce different endoderm populations in different embryonic stem cell populations. It underlies the notion that the evolutionary origin of both cell types is the same and that the pathways evolved for extra-embryonic development in mammals just exploit the ancient modes of germ layer specification that evolved with gastrulation.