Aromatic peptide amphiphiles that self-assemble in response to a trigger, such as pH or enzymes, have the ability to support the culture of somatic cell types, in both two-dimensional (2D) and three-dimensional (3D) culture. Although a fully defined synthetic substrata is required for the successful clinical applications of Embryonic Stem (ES) cells hydrogels of SA aromatic peptide amphiphiles have not been investigated for this purpose. The aim of this investigation is to produce alkaline phosphatase (AP) triggered hydrogels as a substratum for ES cell culture. This SA trigger was chosen as it utilizes inherent biological processes, through the enhanced AP activity of ES cells, with SA occurring in otherwise constant conditions. We also sought to overcome the current inability to consistently control ES cell behaviour in vitro through two different routes that have previously been demonstrated to effect stem cell culture. Firstly, control of the hydrogels mechanical properties and secondly through the incorporation of biological function, principally through the addition of glycosaminoglycans (GAGs). Firstly AP triggered hydrogels of 9-fluorenylmethoxycarbonyl-Tyrosine-OH (Fmoc-Y-OH) were studied and compared with those formed by pH trigger. An unexpected relationship between AP concentration and molecular order was detected. It was observed that the hydrogels stiffness was controlled through the AP concentration; ideal for ES cell culture as Engler et al. (2006) has previously demonstrated the effect material stiffness had on the differentiation pathways chosen by mesenchymal stem cells. Differences between the SA trigger were detected with the hydrogels formed by pH trigger exhibiting significantly lower mechanical properties. This was attributed to the SA process and the disorder that arises from forming all of the hydrogelators instantaneously. The SA process of AP triggered Fmoc-Y-OH showed a 4 stage process. The first stage, dephosphorylation, occurred in a time and AP concentration dependent manner. The second stage transpired due to the spontaneous SA of Fmoc-Y-OH providing a temporary change in fluorenyl environment. The final stages, formation of chiral one dimensional (1D) fibres through proposed -β interactions and gelation through the entanglement of fibres are closely linked. Secondly we investigated AP triggered Fmoc-Phenylalanine-Tyrosine-OH (Fmoc-FY-OH) hydrogels in two distinct physiological environments. When formed in buffer (0.15M, pH 7) an optimum AP concentration was observed in terms of molecular interactions, -β interactions, which translated to the hydrogels mechanical properties. In these conditions helical fibres were imaged by AFM. The second medium for SA was KnockOUT™ DMEM, developed for ES cell culture. Although similarities in the molecular interactions were detected it appears the SA environment effects the structures formed with non-helical fibres imaged. GAGs were successfully incorporated into the hydrogels at biologically relevant levels at the extremes of sulphation. The sulphation patterns of the GAGs secreted by ES cells changes during differentiation and may provide a way to guide cell behaviour through growth factor binding. However the GAGs were not entrapped in the fibre network and leached out into solution limiting their ability to guide ES cell behaviour. Unfortunately both of the hydrogels produced in this study were deemed unsuitable as ES cell substrata due to their instability (Fmoc-Y-OH) or low biocompatibility (Fmoc-FY-OH). However we have demonstrated that it was possible for endogenous AP to trigger SA, indicating that in the future ES cells may be able to form their own substrata preventing the need for exogenous AP.

Although the key role played by stem cells in maintaining hemopoiesis is well recognized, mechanisms that determine stem cell behaviour (e.g. self-renewal) have remained poorly defined. Historical precedent has illustrated the usefulness of in viitro colony assays in facilitating the investigation of various primitive hemopoietic progenitor classes with restricted differentiation and proliferative potential. In particular, such assays have made possible the definition of the sequential nature of a series of events that early erythropoietic cells undergo and suggested properties that might be anticipated to characterize colonies derived from stem cells proliferating and differentiating in vitro. The present studies were undertaken to test the hypothesis that very large, late maturing erythroid colonies previously noted on occasion in routine erythroid colony assays were, in fact, derived from progenitors with the self-renewal and multiple myeloid differentiation properties associated with stem cells. Initial experiments showed that cells capable of yielding macroscopic sized erythroid colonies were present at low frequency in normal marrow but became the predominant erythropoietic cell type after 2 to 3 weeks in flask culture. Macroscopic erythroid colony formation in assays of cells from either source were shown to have: 1) identical very late maturation kinetics (onset of hemoglobinization after 1 week of initial colony growth), 2) the same high erythropoietin requirements, and 3) similar responsiveness to factors present in media conditioned by mitogen stimulated spleen cells. Optimization of these 2 classes of stimulant yielded an assay that was linear down to very low cell concentrations (less than 5 x 103 cells/ml). This made possible the cytological analysis of macroscopic erythroid colonies under conditions where overlap with other colony types was minimal. Such studies revealed that macroscopic colonies contained, in addition to erythroid cells, megakaryocytes ( > 90% of colonies) and granulocyte cells (30% of colonies) including cells of the eosinophil lineage. Such colonies were also found to contain cells capable of macroscopic spleen colony formation in irradiated mice, the conventional assay for mouse hemopoietic stem cells (on average 1 CFU-S per colony of flask culture origin, and 0.3 CFU-S per colony in assays of fresh marrow). Direct evidence for self-renewal was obtained from replating experiments using irradiated feeders to optimize plating efficiency. Mixed colonies of macroscopic size were regularly demonstrable in replating assays after 1, and even 2, generations of mixed colony formation indicating up to 6 self-renewal divisions during colony formation. By comparison to flask culture cells, the extent of self-renewal exhibited by cells in fresh marrow yielding macroscopic erythroid colonies was found to be 5-fold lower. Finally, the in vitro expression of stem cell self-renewal behaviour was investigated in individual colonies. The number of stem cells generated, when assessed either by CFU-S or replating assays was found to vary markedly. This variation was not accounted for by errors of detection or by variations in colony size. Such findings are similar to previous data on the CFU-S content of individual spleen colonies and provide the first evidence that the type of variation in stem cell self-renewal observed in vivo also occurs in vitro where microenvironmental factors are unlikely to be contributing factors. These results are consistent with a model of stem cell self-renewal in which intrinsic cellular factors play the key role in influencing the decision of individual cells to self-renew. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The respiratory system represents a major interface between the body and the external environment. Its design includes a tree-like network of conducting tubules (airways) that carries air to millions of alveoli, where gas exchange occurs. The conducting airways are characterized by their great diversity in epithelial cell types with multiple populations of secretory, multiciliated, and neuroendocrine cells. How these different cell types arise and how these populations are balanced are questions still not well understood. Aberrant patterns of airway epithelial differentiation have been described in various human pulmonary diseases, chronic bronchitis, asthma, neuroendocrine hyperplasia of infancy, and others. The goal of this thesis is to investigate mechanisms of regulation of airway epithelial cell fate in the developing lung epithelium. More specifically, these studies focus on Notch signaling and address a long unresolved issue whether the different Notch ligands (Jagged and Delta) have distinct roles in the epithelial differentiation program of the extrapulmonary and intrapulmonary airways. Moreover, these studies investigate the ontogeny of the bHLH transcription factor Ascl1 and identify its targets in the developing airways as potential regulators of neuroepithelial body (NEB) size and maturation. My studies provide evidence that the Notch ligand families Jag and Dll are required for the specification and formation of different cell lineages in the developing airway epithelia. Jag ligands regulate multiciliated versus secretory (club) cell fates but also controls abundance of basal cell progenitors in extrapulmonary airways. Dll ligands regulate pulmonary neuroendocrine versus club cell fates in intrapulmonary airways. Analysis of mouse mutants showed that loss of Jag ligands has minimal impact on the size or abundance of NEBs and their associated secretory cells while loss of Dll ligands results in an expansion of NEB size and associated cells. To gain additional insights into the potential mechanisms of how neuroendocrine cells develop and undergo aberrant hyperplasia, I characterized the global transcriptional profile of embryonic lungs from mice deficient in Ascl1, which lack NEBs and neuroendocrine cells and identified a number of genes associated with neuroendocrine cell development, maturation, and the NEB microenvironment. Among these genes, components of the catecholamine biosynthesis pathway, such as tyrosine hydroxylase (Th), a key enzyme for catecholamine production, were downregulated in Ascl1 null lungs. Subsequent functional analysis using a pharmacological inhibitor of this pathway in lung organ cultures showed expansion of pulmonary neuroendocrine cells and NEB size, an observation of potential relevance in human diseases in which neuroendocrine cells are aberrantly expanded. Together these studies highlight the distinct role of Notch ligands and further implicate Ascl1 targets, as illustrated by catecholamine pathway components, in regulating epithelial cell fate. Further examination of these pathways may provide insights into the pathogenesis and ultimately therapeutic approaches for airway diseases.

Polychlorinated biphenyls (PCBs) are synthetic persistent organic compounds that are known to be carcinogenic to humans. Changes in telomerase activity and telomere length are hallmarks of aging and carcinogenesis. Retention of telomerase activity and long telomeres are key characteristics of stem cells and progenitor cells. I hypothesize that PCBs modulate telomerase activity and telomeres of hematopoietic stem cells and progenitor cells via interference of gene regulation and potentially disrupt cell differentiation. To investigate this possibility, I used progenitor-like cells, human promyelocytic leukemia cells (HL-60), and stem cells from rat bone marrow. I show that PCB126 and PCB153 display toxic effects on telomerase activity, telomere length and their related gene expression in progenitor-like HL-60 cells, but they did not exert much effect on differentiation. Further, an in vivo/in vitro study using rat bone marrow cells shows that PCB126-induced hematotoxicity, evidenced by reduction in telomerase activity and TERT gene expression, an increase of the differentiation and a change in the differentiation direction towards granulocytes, which indicate an effect on stem cell function. I also show that the most potent dioxin-like congener, PCB126, regulates hTERT gene expression by activation of the AhR pathway. Both AhR and ARNT work together as a repressor of hTERT transcription. This research improves our understanding of mechanisms of PCB126 and PCB153 toxicity on hematopoietic stem cells and progenitor cells, which will ultimately have significant implications for human health.

Introduction Neurogenesis, the development of new neurons, starts soon after the formation of the neural tube and is largely completed by birth. Development of the brain after birth is mainly reliant on the formation of new connections between surviving neurons. However, adult neurogenesis does continue in the subgranular zone of the hippocampus from quiescent adult neural stem cells. Traditionally neural stem cells were cultured as neurospheres, a heterogeneous agglomeration of neural cells at various stages of differentiation. This heterogeneity prevented accurate quantitative analysis. In 2008 Sun et al produced the first non-immortalised human foetal neural stem (NS) cell line from nine week old human foetal cortex. These cells are cultured as monolayers, have a radial glia like appearance, self renew and form all three neural cell types, neurons, astrocytes and oligodendrocytes upon differentiation. More recently human foetal neuroepithelial like (NES) stem cells have been produced from five week old human foetal hind-brain, they resemble neuroepithelial cells, with characteristic rosettes, upon differentiation they appear to form a pure population of neurons. These homogeneous monolayer cultures enable quantitative proteomic analysis, to increase our understanding of early brain development Methods Three NES and two NS cell lines were available for analysis. They proliferate by stimulation from FGF and EGF, removal of these growth factors results in spontaneous differentiation. Proliferating NES and NS cells were compared using SILAC labelling. In addition, each cell line was differentiated for 12 days, 6 timepoints were taken and compared using label free quantitation. Results 4677 proteins were quantitated with 473 differentially expressed, revealing fundamental differences between NES and NS cells. NES cells are less differentiated, expressing SOX2 and LIN28, have active cell cycle processes, DNA elongation, histone modification and miRNA mediated gene silencing. Whereas NS cells are more developmentally defined, express multiple membrane proteins, have activated focal adhesion, thereby increasing their binding and interaction with their environment. NS metabolism is more oxidative, utilises lipid metabolism, the pentose phosphate pathway and produces creatine phosphate. Upon differentiation the cell cycle processes are downregulated and neurogenic and gliogenic processes increased. Conclusion This work represent a detailed in vitro characterisation of non immortalised human foetal neural stem cells, it describes the regulatory, metabolic and structural changes occurring within neural stem cells in early brain development. The information herein points towards de-differentiation potentially through LIN28-let7, as a means to produce more neurogenic neural stem cells in vitro thus aiding regenerative therapies, as well as provides a wealth of information for better understanding neurological developmental disorders.

The physical characteristics (i.e., nanostructure and stiffness) of the extracellular matrix where cells reside have been shown to profoundly affect numerous cellular events in vivo and also been employed to modulate cell behavior in vitro, yet how these physical cues regulate cell behavior is still elusive. Therefore, we engineered a variety of nanotopographies with different shapes and dimensions, and investigated how the nanotopographical cue, through focal adhesions-cytoskeleton-nucleus pathway, affected cell phenotype and function. We further designed and fabricated well-defined substrates which had either identical biochemical cue (adhesive ligand presentation) but different nanotopographical cues or identical nanotopography but different biochemical cues, and dissected the roles of these cues in cell modulation. In addition, we revealed that the human mesenchymal stem cells (hMSCs) could obtain nanotopographical memory from the past culture environment, and the nanotopographical memory influenced the future fate decision of the hMSCs. Moreover, we evaluated the effects of substrate nanotopographical and stiffness cues on the fibrogenesis of human lung fibroblasts in response to carbon nanotubes and highlighted the significance of these physical cues in the development of physiologically relevant in vitro models for nanotoxicological study. The mechanistic understanding of the physical regulation of cell behaviors will provide important insight into the advancement of cell culture technologies and the recreation of biomimetic in vitro tissue/organ models.

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

Isocitrate Dehydrogenase-1 has been found to be mutated in over 70% of lower grade gliomas and has become an important diagnostic tool for tumor prognosis, however its role in glioma development, and its impact on response to therapy, is still not fully understood. Unmutated IDH1 functions to convert isocitrate to alpha-ketoglutarate (a-KG) in the tricarboxylic acid (TCA) cycle. Mutated IDH1R132H changes the enzymatic equilibrium and converts a-KG to 2-hydroxyglutarate (2-HG), an oncometabolite. IDH1R132H mutated tumors show an elevated production of 2-HG and epigenetic alterations in DNA and histone methylation. This mutation is predominantly seen in the proneural glioma subtype in which oligodendrocytes progenitors (OPCs) are considered the cell of origin due to phenotypic similarities. The effect of IDH1R132H mutation in cellular transformation has not been fully established. Epigenetic modifications connect genotype to phenotype by genetic expression alterations and epigenetic modifications are necessary for OPC differentiation. Tri-methylation of lysine residue K27 on histone H3 (H3K27me3) is a repressive mark associated with cell pluripotency. H3K27me3 is trimethylated by Enhancer of Zeste 2 (EZH2) and demethylated by the a-KG-dependent demethylases UTX/KMD6A and JMJD3/KDM6B. 2-HG is a competitive inhibitor of a-KG-dependent demethylases, providing a mechanistic link between IDH mutations and increases H3K27me3 by inhibiting demethylation. In this thesis, we evaluated the epigenetic changes in mouse models of IDH mutant and wildtype glioma and genetically-transformed OPCs and tested the effects of drugs that target specific epigenetic marks. We developed a mouse model of glioma to compare IDH1R132H cells to wildtype glioma cells and found that although there was no difference in survival, IDH1R132H gliomas have increased levels of 2-HG by MALDI-IMS and metabolomic analysis. Interestingly, based on RNA-sequencing analysis our IDH1R132H model has a more OPC-restricted expression profile compared the wildtype glioma model which have higher enrichment of genes from other cell lineages, including neurons, astrocytes, myelinating oligodendrocytes and microglia. We used the EZH2 inhibitor (Tazemetostat, EPZ-6438) and found that this treatment was not cytotoxic or cytostatic to our cells although H3K27me3 was reduced. Interestingly, Tazemetostat treatment increased the expression of non-OPC genes (genes normally expressed by other lineages as assessed using the Barres transcriptomic database). To better understand how IDH1R132H influences OPC transformation, we transformed OPCs in vitro. OPCs were isolated from floxed p53 postnatal day 5 mice from and retrovirally infected with viruses to delete p53 alone or to also express IDH1R132H. OPCs that express IDH1R132H had increased levels of 2-HG by metabolomics and showed alteration in H2K27 methylation and acetylation that resembled those seen in glioma cells. Standard methods of western blot analysis consist of analyzing whole cell lysate, cytoplasmic and nuclear fractionation, or histone acid extraction. To analyze both the cytoplasmic fraction as well as histone modification, I developed a cellular extraction method in which cells were fractionated and the nuclear fraction was acid extracted. This method allows for the analysis of both cytoplasmic proteins as well as histone modifications by western blot. Using this method, we found that treating glioma cells or OPCs with synthetic cell permeable octyl-2HG, or expressing IDH1R132H, caused cells to have increased H3K27me3, while treatment with Tazemetostat caused a decrease in H3K27me3. Based on the RNA-sequencing data we found that increased H3K27me3 (ID1R132H mutation) express more OPC-like phenotype while reduced H3K27me3 (Tazemetostat treatment) induced an upregulation of genes associated with other lineages making them less restricted to the OPC transcriptional phenotype. We found that in both the glioma cells and OPCs, Tazemetostat treatment decreased H3K27me3 and increased H3K27ac. Based on the increase of H3K27ac after Tazemetostat treatment, we hypothesized that a Histone deacetylase inhibitor (HDACi) would be synergistic. We found that although the HDACi Panobinostat was less cytotoxic to IDH1R132H mutated glioma cells and OPCs, co-treatment with Tazemetostat is synergistic in mutant and wildtype models. We also saw that in IDH1R132H ex vivo slices, the co-treatment reduced tumor marker composition. These findings point to a novel therapeutic strategy for IDH1-mutated proneural gliomas that targets the specific epigenetic alteration in these tumors.

Cardiac stem cells (CSCs)-based therapy provides a promising avenue for the management of ischemic heart diseases. However, engrafted CSCs are subjected to acute cell apoptosis in the ischemic microenvironment. Here, stem cell antigen 1 positive (Sca-1+) CSCs proved to own therapy potential were cultured and treated with H2O2 to mimic the ischemia situation. As autophagy inhibitor, 3-methyladenine (3MA), inhibited H2O2-induced CSCs apoptosis, thus we demonstrated that H2O2 induced autophagy-dependent apoptosis in CSCs, and continued to find key proteins responsible for the crosstalk between autophagy and apoptosis. Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), increased upon cardiomyocyte injury with unknown functions in CSCs, was increased by H2O2. NR4A2 siRNA attenuated H2O2 induced autophagy and apoptosis in CSCs, which suggested an important role of NR4A2 in CSCs survival in ischemia conditions. Reactive oxygen species (ROS) and NF- κB (P65) subunit were both increased by H2O2. Either the ROS scavenger, N-acetyl-lcysteine (NAC) or NF-κB signaling inhibitor, bay11-7082 could attenuate H2O2-induced autophagy and apoptosis in CSCs, which suggested they were involved in this process. Furthermore, NAC inhibited NF-κB activities, while bay11-7082 inhibited NR4A2 expression, which revealed a ROS/NF-κB/NR4A2 pathway responsible for H2O2- induced autophagy and apoptosis in CSCs. Our study supports a new clue enhancing the survival rate of CSCs in the infarcted myocardium for cell therapy in ischemic cardiomyopathy.

Cellular cardiomyoplasty is a cell therapy using stem cells or progenitor cells for myocardial regeneration to improve cardiac function and mitigate heart failure. Since we first published cellular cardiomyoplasty in 1989, this procedure became the innovative method to treat damaged myocardium other than heart transplantation. A significant improvement in cardiac function, metabolism, and perfusion is generally observed in experimental and clinical studies, but the improvement is mild and incomplete. Although safety, feasibility, and efficacy have been well documented for the procedure, the beneficial mechanisms remain unclear and optimization of the procedure requires further study. This chapter briefly reviews the stem cells used for cellular cardiomyoplasty and their clinical outcomes with possible improvements in future studies.