Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. I present a novel, single-step method of generating murine ESC (mESC)-derived chondrocytes in monolayer cultures in chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, Activin A or Wnt3a. Prolonged culture with sustained Activin A, TGFβ3 or BMP4 supplementation led to robust chondrogenic induction. A short pulse of Activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with Activin A or with Activin A followed by TGFβ3 may specifically promote articular cartilage formation. Thus, I devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. I am also in the process of creating a Col2a1 promoter-controlled, Cre-inducible reporter cell line to be used in my SF culture system using the Multisite Gateway® cloning technology. ESCs undergoing chondrogenic differentiation can be identified and quantified in HTS via the expression of fluorescent proteins. In addition, this transgenic line can be used to isolate ESC-derived chondrocytes as well as their progeny via cell sorting or antibiotic selection for in-depth characterization. The modular design of my construct system allows transgenic lines to be generated using various promoters of chondrogenic marker genes to perform parallel HTS analyses.

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain brings promise for the treatment of neurological diseases and has yielded new insight into brain cancer. The complete repertoire of signaling pathways that governs these cells however remains largely uncharacterized. This thesis describes how chemical genetic approaches can be used to probe and better define the operational circuitry of the NSC. I describe the development of a small molecule chemical genetic screen of NSCs that uncovered an unappreciated precursor role of a number of neurotransmitter pathways commonly thought to operate primarily in the mature central nervous system (CNS). Given the similarities between stem cells and cancer, I then translated this knowledge to demonstrate that these neurotransmitter regulatory effects are also conserved within cultures of cancer stem cells. I then provide experimental and epidemiologically support for this hypothesis and suggest that neurotransmitter signals may also regulate the expansion of precursor cells that drive tumor growth in the brain. Specifically, I first evaluate the effects of neurochemicals in mouse models of brain tumors. I then outline a retrospective meta-analysis of brain tumor incidence rates in psychiatric patients presumed to be chronically taking neuromodulators similar to those identified in the initial screen. Lastly, by further exploring the phenotype and function of neurotransmitter pathways in purified populations of human NSCs, I determined that neurotransmitter pathway gene expression exists in a functionally heterogeneous phase-varying state that restricts the responsiveness of these populations to various stimuli. Taken together, this research provides novel insights into the phenotypic and functional landscape of neurotransmitter pathways in both normal and cancer-derived NSCs. In additional to a better fundamental understanding of NSC biology, these results suggest how clinically approved neuromodulators can be used to remodel the mature CNS and find application in the treatment of brain cancer.

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain brings promise for the treatment of neurological diseases and has yielded new insight into brain cancer. The complete repertoire of signaling pathways that governs these cells however remains largely uncharacterized. This thesis describes how chemical genetic approaches can be used to probe and better define the operational circuitry of the NSC. I describe the development of a small molecule chemical genetic screen of NSCs that uncovered an unappreciated precursor role of a number of neurotransmitter pathways commonly thought to operate primarily in the mature central nervous system (CNS). Given the similarities between stem cells and cancer, I then translated this knowledge to demonstrate that these neurotransmitter regulatory effects are also conserved within cultures of cancer stem cells. I then provide experimental and epidemiologically support for this hypothesis and suggest that neurotransmitter signals may also regulate the expansion of precursor cells that drive tumor growth in the brain. Specifically, I first evaluate the effects of neurochemicals in mouse models of brain tumors. I then outline a retrospective meta-analysis of brain tumor incidence rates in psychiatric patients presumed to be chronically taking neuromodulators similar to those identified in the initial screen. Lastly, by further exploring the phenotype and function of neurotransmitter pathways in purified populations of human NSCs, I determined that neurotransmitter pathway gene expression exists in a functionally heterogeneous phase-varying state that restricts the responsiveness of these populations to various stimuli. Taken together, this research provides novel insights into the phenotypic and functional landscape of neurotransmitter pathways in both normal and cancer-derived NSCs. In additional to a better fundamental understanding of NSC biology, these results suggest how clinically approved neuromodulators can be used to remodel the mature CNS and find application in the treatment of brain cancer.

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. I present a novel, single-step method of generating murine ESC (mESC)-derived chondrocytes in monolayer cultures in chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, Activin A or Wnt3a. Prolonged culture with sustained Activin A, TGFβ3 or BMP4 supplementation led to robust chondrogenic induction. A short pulse of Activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with Activin A or with Activin A followed by TGFβ3 may specifically promote articular cartilage formation. Thus, I devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. I am also in the process of creating a Col2a1 promoter-controlled, Cre-inducible reporter cell line to be used in my SF culture system using the Multisite Gateway® cloning technology. ESCs undergoing chondrogenic differentiation can be identified and quantified in HTS via the expression of fluorescent proteins. In addition, this transgenic line can be used to isolate ESC-derived chondrocytes as well as their progeny via cell sorting or antibiotic selection for in-depth characterization. The modular design of my construct system allows transgenic lines to be generated using various promoters of chondrogenic marker genes to perform parallel HTS analyses.

The ability to generate cardiomyocytes from either embryonic stem cells or induced pluripotent stem cells provides an unprecedented opportunity to establish human in vitro models of cardiovascular disease as well as to develop platforms for the testing of novel cardiac therapeutics. We designed two different platforms, a biowire platform and post deflection platform, to generate engineered heart tissues (EHTs) to study a fundamental process in cardiomyocytes: hypertrophy. Both pathological and physiological hypertrophy was studied in order to garner a better understanding of each process. Physiological hypertrophy characteristics were observed using the biowire platform seen in improved myofibril alignment and downregulation of fetal genes. When electrical stimulation was added, a rate dependent effect on sarcomere maturation was observed by the increased frequency of I-bands and H-zones. Certain hallmark features of pathological hypertrophy, such as upregulation of brain natriuretic peptide and sarcomere structure breakdown, were recapitulated when EHTs were treated with isoproterenol.

No description available.

No description available.

The ability to generate cardiomyocytes from either embryonic stem cells or induced pluripotent stem cells provides an unprecedented opportunity to establish human in vitro models of cardiovascular disease as well as to develop platforms for the testing of novel cardiac therapeutics. We designed two different platforms, a biowire platform and post deflection platform, to generate engineered heart tissues (EHTs) to study a fundamental process in cardiomyocytes: hypertrophy. Both pathological and physiological hypertrophy was studied in order to garner a better understanding of each process. Physiological hypertrophy characteristics were observed using the biowire platform seen in improved myofibril alignment and downregulation of fetal genes. When electrical stimulation was added, a rate dependent effect on sarcomere maturation was observed by the increased frequency of I-bands and H-zones. Certain hallmark features of pathological hypertrophy, such as upregulation of brain natriuretic peptide and sarcomere structure breakdown, were recapitulated when EHTs were treated with isoproterenol.

miRNAs are short, non-coding RNA molecules that regulate gene expression posttranscriptionally through inhibition of translation and/or mRNA degradation. Mammalian development is a complex series of developmental transitions, which relies on accurate spatial and temporal regulation of gene expression and we are interested in the role that miRNAs may play in these developmental transitions. An initial objective was to establish which, if any, miRNAs were dynamically regulated in a cell model of an early developmental transition, and to establish whether differential expression of any particular miRNA played a functional role in this developmental process. Having established a role for specific miRNAs, further objectives were to assess the reliability of current miRNA-mRNA target identification procedures and to assess the general role of miRNAs in cellular differentiation. In order to explore the roles of miRNAs during an early developmental transition, an embryonic stem (ES) cell model of trophectoderm differentiation was used. In this model system the expression of the key ES cell regulatory gene, Oct4, can be conditionally repressed, which induces the ES cells to differentiate down the trophectoderm lineage. The expression of microRNAs was profiled in this model system by cloning and sequencing of small RNAs. This approach identified miRNAs that were dynamically regulated during differentiation. The expression patterns of differentially regulated miRNAs were confirmed by miRNA northern analysis. The miRNA profiling data showed that mmu-miR-294 and mmu-mir-295 are expressed at similar levels in ES cells and differentiated cells, which disagrees with previous reports that these miRNAs are ES cell specific. Several of the miRNAs with higher expression levels in differentiated cells are encoded within a placental-enriched polycomb group gene, Sfmbt2, suggesting an important role for these miRNAs in extraembryonic development. One of the miRNAs that was expressed at higher levels in ES cells than in differentiated cells, mmu-miR-92a, was shown to play a role in regulation of cell proliferation. Three current methods of identifying miRNA targets were assessed. A sequencebased method using the web-based utility miRecords, which amalgamates results from numerous target prediction databases, was used to generate lists of potential targets of the Sfmbt2 miRNA cluster and of mmu-miR-92a. Amalgamating results from multiple target prediction programs may improve the likelihood that the predicted targets are real. Exemplifying this, the single mmu-miR-92a target that was predicted by six different target prediction programs had been previously experimentally verified. An experimental method of identifying direct miRNA targets, PAR-CLIP, was investigated but proved technically limiting for routine use in the laboratory. A proteome-based experimental method for identifying potential miRNA targets, called SILAC, was successfully used to identify proteins that were differentially expressed in the cell model of trophectoderm differentiation. Differential expression of two of these proteins, CTBP2 and CKB, was confirmed by western analysis. miRecords was then used to assess whether the differentially expressed proteins were likely to be targets of the differentially expressed miRNAs that had been identified in the miRNA profiling analysis. The general role of miRNAs in cell differentiation was investigated using a cell line that does not express miRNAs. This ES cell line is deficient for the miRNAprocessing enzyme DGCR8, which results in loss of expression of mature miRNAs in these cells. Compared to wild type ES cells, miRNA-deficient ES cells expressed normal levels of the ES cell marker genes Oct4 and Sox2 but elevated levels of Nanog. In contrast to wild type ES cells, miRNA-deficient ES cells did not upregulate the mesoderm marker gene Brachyury during embryoid body differentiation and showed reduced upregulation of the endoderm marker gene Gata6. These findings suggest that miRNAs are not required for maintenance of pluripotency, but are essential for proper ES cell differentiation. The results presented in this thesis show that miRNAs are dynamically expressed during a mammalian developmental transition and are involved in regulating early developmental processes. We believe that miRNAs act as an additional level of genetic regulation to ensure canalisation during embryonic development.

Pluripotent stem cells (PSCs) can self-renew indefinitely in culture while maintaining their capacity to differentiate into any cell type of an organism, thus offering novel sources for drug screening, in vitro disease modelling, and cell replacement therapies. However, due to their sensitive nature, many PSC lines are still cultured using undefined components such as serum or serum-derived components, on either feeder cells or complex protein mixes such as Matrigel or gelatine. In order to fully realize the potential of these cells we need controlled, completely defined and xeno-free culturing conditions that maintain growth and survival of homogenous, non-differentiated colonies. This thesis focuses on the in vitro maintenance of both mouse and human PSCs, analysing the media and substrate requirements of these cells and linking them to the intracellular signalling pathways involved in the maintenance of pluripotency and self-renewal. Benchmarking of commercially available culture methods for PSCs has been performed, evaluating their capacity to maintain pluripotency and growth of undifferentiated PSCs over several passages and reporting new characteristics, like the tendency of mouse PSCs to grow as floating spheres in 2i medium, a novel media formulation that uses two inhibitors to hinder differentiation capacity and subsequently induce pure, undifferentiated cultures. The major finding in this thesis is the identification of Inter-α-Inhibitor (IαI) as a protein able to activate the previously described signal-transduction pathway Yes/YAP/TEAD in mouse PSCs and to induce transcription of the well-known stem cell transcription factors Nanog and Oct3/4. IαI is a serum protein found in high concentration in human serum that had been traditionally described as an extracellular matrix remodelling protein. For the first time, we describe IαI to have signalling capacity on PSCs. Moreover, IαI is demonstrated to induce attachment, growth and long-term survival of undifferentiated mouse and human PSCs when added to serum-free, chemically defined media. IαI is the first molecule described to date to induce attachment of human PSCs on uncoated, standard tissue-culture treated plastic, just by supplementation as a soluble molecule at the seeding step. Following this discovery, we evaluate a novel culture method using the completely defined, serum-free E8 medium supplemented with IαI (E8:IαI) for long-term propagation of four different human PSC lines and discover that IαI can indeed support long-term culture with maintained pluripotency, differentiation capacity, growth rate and genetic stability. Moreover, in contrast to the control culture method using a commercially available surface coating, IαI supplementation can support single cell passaging of human PSCs, and adapt feeder-dependent cultured human PSCs to E8:IαI with high efficiency. A mouse PSC line is also grown for over 20 passages in IαI with retained pluripotency, differentiation capacity and genetic stability. IαI is inexpensive to produce and derived from human plasma, and could therefore be produced in compliance with Good Manufacturing Practices. Ultimately, our group aims to develop and test large-scale, completely defined, xeno-free culturing methods for PSCs, suitable for pharmacological and medical applications.