Thesis (Ph. D.)--University of Nevada, Reno, 2008. / "May, 2008." Includes bibliographical references (leaves 117-133). Online version available on the World Wide Web.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases, in part by modulating adaptive and innate immune responses. Since metabolism governs the phenotype and function of immune cells, the aim of this thesis was to investigate whether NSCs have the ability to regulate the immunometabolic components underpinning neuroinflammation. Herein I have identified a new mechanism by which transplanted somatic and directly-induced NSCs counteract CNS-compartmentalised chronic inflammation in mice. NSC transplantation reduces the immunometabolite succinate in the cerebrospinal fluid, while decreasing the burden of mononuclear phagocyte (MP) infiltration and secondary CNS damage. Mechanistically, the anti-inflammatory activity of NSCs arises in response to succinate released by inflammatory MPs, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, thus initiating prostaglandin E2 secretion and extracellular succinate scavenging. This work uncovers a succinate-SUCNR1 axis in NSCs that clarifies how stem cells respond to inflammatory metabolic signals to inhibit the activation of pro-inflammatory MPs in the chronically inflamed brain.

Mesoderm is one of the three primary germ layers from which the cardiovascular system, muscle and bone originate and derivatives of the mesoderm lineage are affected in a number of pathologies. Therefore, understanding the mechanisms regulating formation of mesoderm is interesting for a diversity of diseases and clinical application. In vivo study of human development beyond gastrulation is technically challenging and the mechanisms controlling mesoderm specification are difficult to study since the maximum number of days allowed to grow human embryos is 14 days. Thus, in this dissertation I use human pluripotent stem cells (hPSCs) as a simplified model of human development. Studies have shown that the cell cycle machinery plays a direct role in the differentiation of endoderm and ectoderm lineages but its role in guiding mesoderm subtype formation remains elusive. In this dissertation, I provide new insights of the importance of the cell cycle regulators in mesoderm specification. I first developed tools such as the FUCCI2A reporter line to isolate cells in the different cell cycle phases and to investigate propensity of mesoderm differentiation. I have shown that the propensity of differentiation into the three mesoderm subtypes lateral plate mesoderm, cardiac mesoderm and presomitic mesoderm varies during the cell cycle phases, with differentiation being more efficient in the G1 and to a lesser extend in G2/M phase. Furthermore, I developed a protocol where cells can be efficiently synchronised in the different cell cycle phases using the G2/M inhibitor nocodazole. Using this tool, I showed that developmental signalling pathways such as BMP and WNT are active in all cell cycle phases indicating that alternative mechanisms are involved in the differentiation process. In order to further explore these mechanisms, I investigated the role of cell cycle regulators controlling the G1 and G2 checkpoint. I have shown that the cell cycle regulators CDK4/6, CDK2, Retinoblastoma phosphorylation and CDK1 are essential for mesoderm subtype formation. Furthermore, I have shown that CDK1 regulates the activity of ERK1/2 signalling, an important pathway for the differentiation process confirming the existence of complex interplays between cell cycle machinery, signalling pathways and transcription factors in mesoderm subtype formation. This knowledge will be useful to further improve protocols for generating mesoderm subtypes from hPSCs for clinical applications such as drug screening, disease modelling and cell based therapy.

M. Tech. / Stem cells are regarded as undifferentiated cells that are capable of selfrenewal, proliferation, production of a great number of differentiated progeny, and regeneration of tissues (Blau et al., 2001). The therapeutic potential of multilineage stem cells for tissue engineering (TE) applications is vast. Two general types of stem cells are potentially useful for this application: embryonic stem cells (ESCs) and adult (autologous) stem cells (Zuk et al., 2001). Traditionally, ESCs are isolated from the inner cell mass (ICM) of blastocysts, however harvesting of these cells results in the death of the embryo, which has led to ethical, religious and political issues (Moore, 2007). In contrast, adult stem cells, by virtue of their nature, are immunocompatible and have no ethical issues associated with their use (Zuk et al., 2001). Subcutaneous adipose tissue is an active and highly complex tissue composed of several different cell types, and is derived from the mesodermal germ layer and contains a supportive stromal vascular fraction (SVF) that can be easily isolated. This SVF contains a heterogeneous mixture of cells including preadipocytes (Raposio et al., 2007; Schäffler and Büchler, 2007; Jurgens et al., 2008). The preadipoctyes are considered as the multipotent stem cells termed adipose derived stem cells (ADSCs) that have similar properties to bone marrow mesenchymal stem cells (BM-MSCs) (Fraser et al., 2006). ADSCs are idyllic for cellular therapy applications due to various factors: they can be harvested, multiplied and handled easily, efficiently and noninvasively, they have a pluripotential and proliferative capacity comparable to BM-MSCs, and morbidity to donors is considerably less, requiring only local anaesthesia and a short wound healing time. Human ADSCs (hADSCs) can be expanded in an undifferentiated state and have multipotential differentiation capacity along the classical mesenchymal lineages of adipogenesis, osteogenesis, chondrogenesis and myogenesis (de Villiers et al., 2009).

No description available.

Both cDNA tag-based and DNA chip hybridization assays have revealed widespread transcriptional activity across mammalian genomes, providing a rich source of novel protein-coding and non-coding transcripts. Annotation and functional evaluation of this undefined transcriptome space represents a major step towards the comprehensive definition of biomolecules regulating the properties of living cells, including embryonic stem cells (ESCs) and their derivatives. In this study I analysed 87 rare mRNA transcripts from human ESCs that mapped uniquely to the human genome, in regions lacking evidence for known genes or transcripts. In addition, the transcripts appeared enriched in the hESC transcriptome as enumerated by serial analysis of gene expression (SAGE). Full-length transcripts corresponding to twelve novel LongSAGE tags were recovered and evaluated with respect to gene structure, protein-coding potential, and gene regulatory features. In addition, transcript abundance was compared between RNA isolated from undifferentiated hESCs and differentiated cells. Analysis of full-length transcripts revealed that the novel ORFs did not exceed a size of 129 amino acids and no matches were observed to well characterized protein domains. Interesting protein level predictions included small disulfide-bonded proteins, known members of which are important in a variety of biological processes. Transcripts evaluated for differential expression by real-time RT-qPCR (Reverse Transcription followed by real-time quantitative Polymerase Chain Reaction) were found to be variably expressed (0.2- to 4.5-fold) in Day-2 or Day-4 retinoic acid-induced differentiation cultures compared to undifferentiated hESCs. Relative quantitation using a universal reference RNA (derived from pooled adult tissues) showed large differences in novel transcript levels (0.002- to 35-fold) compared to hESCs. Collectively, these results provide a detailed analysis of a set of novel hESC transcripts and their abundance in early and adult differentiated cell types, both of which may advance our understanding of the transcriptional events governing stem cell behavior. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Polycomb proteins are epigenetic regulators that are critical in mediating gene repression at critical stages during development. Core and accessory proteins make up the Polycomb Repressive Complex 2 (PRC2), which is responsible for trimethylation of lysine 27 on histone 3 (H3K27me3), leading to maintenance of chromatin compaction and sustained gene repression. Classically, Polycomb accessory proteins are often thought of as having minor roles in fine-tuning the repressive action of PRC2. Their actions have often been attributed to chromatin recognition, targeting to specific loci and enhancing methyltransferase activity. In our previous work in mouse embryonic stem cells (ESCs), we showed that Polycomb-like 2 (Mtf2/Pcl2) is critical for PRC2-mediated regulation of stem cell self-renewal through feed-forward control of the pluripotency network. In moving beyond the ESC model system, we sought to interrogate the role of Mtf2 in vivo by creating a gene-targeted knockout mouse model. Surprisingly, we discovered a tissue-specific role for Mtf2 in controlling erythroid maturation and hematopoietic stem cell self-renewal. Via its regulation of other PRC2 members, Mtf2 is critical for global H3K27me3 methylation at promoter-proximal sites in developing erythroblasts. Thus, Mtf2 is required for proper maturation of erythroblasts. Loss of Mtf2 also reduces HSC self-renewal leading to stem cell pool exhaustion. Additionally, misregulation of Mtf2 in leukemia models contributes to massive leukemic blast expansion at the expense of leukemic stem cell self-renewal. In the developing hematopoietic system, Mtf2 functions as a core complex member, controlling epigenetic regulation of self-renewal and maturation of both stem and committed cells.

Basal cells (BCs) are multipotent tissue-specific stem cells of a variety of organs including the skin, digestive and respiratory tract. BCs are broadly identified by expression of Krt5, Krt14 and the transcription factor p63. In the adult airways, BCs are not only important for normal maintenance but also crucial for epithelial repair after injury. However, the embryonic origin of these adult stem cells remains elusive. Previous reports showed that p63+ cells appear early during airway development, but these do not express markers of adult BCs, raising the question whether these cells represent BC precursors. Moreover, little was known whether embryonic BCs have an impact in the adult pool of progenitors that mediate responses of the lung to injury or pulmonary diseases. The goal of this thesis is to address these gaps of knowledge using a variety of technologies, including functional and lineage tracing analysis in vivo in mouse genetic models, injury modeling, high-throughput profiling and gene regulation approaches. This thesis is to comprehensively characterize airway BCs in development, injury-repair, and homeostasis. These studies revealed a previously unrecognized broader role of embryonic p63+ cells in the establishment of the stem cell pools of the lung pre and postnatally. Surprisingly, lineage analysis showed that early in development these cells were able to generate all epithelial cell types of the airways and alveolar compartment. However, as development proceeds, they underwent two sequential lineage segregation events to finally generate two regionally distinct adult stem cell pools. One of these became the well-known BCs that populate extrapulmonary airways through an undescribed maturation process from the perinatal stage to adulthood, and the other was identified as a rare stem cell pool in the pseudostratified epithelium of intrapulmonary airways which maintained immature and quiescent throughout lifetime. Moreover, the latter responded uniquely to lung injury induced by H1N1 viral infection. Recent studies have demonstrated that BC-like p63+ Krt5+ cell clusters (“Krt5+ pods”) are ectopically present in the areas of severe alveolar injury by H1N1 viral infection. The presence of these pods has been associated with pathological scars in several human pulmonary diseases including idiopathic pulmonary fibrosis (IPF) and acute respiratory distress syndrome (ARDS). However, their cellular origin has been intensely debated. This thesis showed that this rare progenitor pool is established during embryonic development when airways are still branching. Further characterization demonstrated a p63 gene dosage dependency in the specification/maintenance of this rare progenitor pool. By utilizing multiple lineage-tracing lines, an underappreciated diversity of this pool was revealed by showing a novel subpopulation carrying secretory lineage marker spatially restricted to intrapulmonary airways. Further molecular characterization and genetic manipulation of this rare progenitor pool may provide valuable cues to understand the pathogenesis about pulmonary disorders and to develop effective therapies. Moreover, the molecular signatures of tracheal embryonic E18.5 preBCs and adult TrBCs were generated through high-throughput profiling, which provided hints about the genetic regulation of airway BC maturation process and generated potential molecular landmarks for the in vitro ES/iPS cell differentiation towards airway BCs. In addition, single cell RNA-sequencing analyses revealed heterogeneity of adult BCs in the tracheal and esophageal epithelia. Lastly, candidate master regulators of their differentiation programs in homeostatic and metaplastic states were identified through unbiased systems biology algorithms, which will be further validated in functional assays in the near future. Taken together, the studies in this thesis comprehensively characterized airway BCs in development, injury-repair and homeostasis. This thesis work showed the newly identified p63+ airway progenitors before E10.5 are multipotent for all lung epithelial lineages and this multipotency gets restricted to proximal fate at E10.5. In the adult injury-repair, this thesis work for the first time revealed that the H1N1-induced Krt5+ pods are generated by bronchial p63+ Krt5- progenitors, which originate from a subpopulation of E13.5 intrapulmonary p63+ progenitors. At homeostasis, this thesis work uncovered a previously underappreciated heterogeneity of BCs in both airways and esophagus, and provided molecular foundations for further explorations into the mechanistic perspectives of BC cellular identity maintenance.

Tumour initiation and progression is thought to be driven by a small population of tumor initiating cells (TICs) or cancer stem cells (CSCs), which have the capacity to migrate and cause metastases and contribute to tumour relapse. These cells possess properties that are similar to those of normal tissue stem cells, which include the capacity to undergo self-renewal as well as the capacity to give rise to more differentiated progenitor cells, which comprise the bulk of the tumour cell population. Thus far, the clinical significance of these cells in breast cancers has not been extensively explored with regard to their relationship with tumour pathology or patient survival. In this thesis we evaluate the presence of these cells in terms of clinicopathological tumour characteristics and patient outcome, as well as assess potential markers of breast CSCs for prognostic significance. Through the quantification of breast CSCs in primary breast tumours using in vivo xenografts assays we show that their presence correlates with aggressive tumour characteristics. In addition, we propose that markers of breast CSCs may differ based on the molecular subtype of the tumour, and that these markers have prognostic significance in patients. / Thesis / Master of Science (MSc)

Wnt signaling regulates critical cellular interactions throughout normal development and directs cell fate decisions of stem cells. Previous work by our lab implicates -catenin as an essential modulator of embryonic stem cell self-renewal and differentiation. Genetic studies in mice have demonstrated broad functional redundancies between the most downstream effectors of the Wnt signaling cascade, the T-cell factor / Lymphoid enhancer factor (TCF/LEF) family of transcription factors. Despite this, loss-of-function experiments suggest that -catenin reinforces the pluripotent state by mediating a TCF switch in which repressive TCF7L1 is replaced with activating TCF7. However, these experiments do not account for potential confounding functional compensation by other TCF/LEF factors. As such, I hypothesized that TCF7 and TCF7L1 are functionally redundant in mouse embryonic stem cells and bind a largely overlapping set of target genes and interacting proteins. In support of this notion, we demonstrated that both TCF7 and TCF7L1 were similarly able to restore the altered transcriptomic profile and differentiation deficits observed in mouse embryonic stem cells (mESCs) lacking all full-length TCF/LEFs. With the expectation that TCF7 and TCF7L1 recruit similar transcriptional co-regulators to a broadly overlapping set of target genes, we employed the unbiased techniques, ChIP-seq and BioID to test our hypothesis. We observed that regardless of the degree of Wnt signaling activity, TCF7L1 was more abundantly associated with chromatin than TCF7, and TCF7 and TCF7L1 regulate distinct target genes. We demonstrated that Wnt stimulation, simulated by GSK-3 inhibition, facilitates TCF7L1 interactions with transcriptional modulators such as the BAF and nuclear receptor co-repressor complexes, despite a reduction in TCF7L1 levels. Taken together, the work in this thesis provides new insights into the mechanisms of Wnt target gene regulation by the TCF/LEF factors. / Thesis / Doctor of Philosophy (PhD) / Stem cells are capable of giving rise to multiple different cell types and thus are able to generate all adult tissues. The identity of a cell is controlled by external signals that regulate internal programs encoded by our genes. The execution of the instructions in genetic programs is conducted by proteins called transcription factors that can turn different genes on or off, giving rise to distinct cell types. The T-Cell Factors and Lymphoid Enhancer Factor (TCF/LEFs) are a family of four transcription factors regulated by external signaling molecules called Wnts. By using the TCF/LEFs, Wnts establish gene outputs that determine the identity of cells throughout embryonic development and in adult tissues. However, the mechanisms used by this family of transcription factors to establish the programs controlling cellular identity remain poorly understood. Using genetically engineered mouse embryonic stem cells, we have uncovered new information about the mechanisms TCF/LEFs use to regulate gene function, identified programs controlled by TCF/LEFs, and discovered potential protein partners that work with TCF/LEFs to implement genetic programs. This thesis provides novel insights into the control of cell identity by the TCF/LEFs, which has implications for the numerous human diseases linked to abnormal Wnt-mediated signaling.