Work in our lab has resulted in the development of a novel approach to creating a more developmentally plastic human dermal fibroblast (hDF) phenotype that allows for the study of molecular mechanisms involved in cell-fate conversion. Culturing hDF under defined culture conditions (5% O2 and supplementation with fibroblast growth factor FGF2) induces induced the regeneration competent (iRC) phenotype that is characterized by stem cell gene expression, and increased life-span in vitro. The work presented in this thesis further characterizes the system, and describes an overall shift in extracellular matrix and adhesion molecules in human dermal fibroblasts (hDF) undergoing the transition to a more developmentally plastic phenotype (iRC). This work suggests that we create the initiation phase of Mesenchymal-to-Epithelial Transition (MET) during conversion to the iRC phenotype. This transition is marked by loss of integrin alpha-11 (α11) and its binding partner Collagen-I (COL-I). Moreover, we describe the mechanism for the down-regulation of α11 that is mediated by FGF2 activation of ERK1/2 through systematic investigation of several potential molecular mechanisms. The body of work presented here shows that the ERK 1/2 mediated down-regulation of α11 is independent of activation of TGF-β1-mediated regulation of α11. In addition to down-regulation of α11, an overall shift in the transcript levels of other adhesion molecules is observed, which demonstrates that iRC are most likely transitioning their attachment to a laminin and fibronectin-based matrix. These results suggest that iRC may be producing a more “pro-regenerative matrixâ€�. We hypothesize that the changes in integrin expression profile and interaction with ECM serve as a feedback loop during the iRC phenotype shift. Our findings suggest that this “pro-regenerativeâ€� shift in attachment of iRC as well as the ERK 1/2 mediated down-regulation of α11 could be exploited in wound healing biology and fibrosis research. Manipulation of the dynamic relationship between TGF-β1 and FGF2 has the potential to reduce scar deposition. Further identification of molecular mechanisms controlling this phenotype conversion will allow development of strategies for in situ manipulation of wound healing outcomes.

Human embryonic stem cells (hESCs) can provide a unique approach for novel tissue engineering applications. Previous groups have shown that hESCs can differentiate into specialized cell types through the generation of human embryoid bodies (hEBs). These multi-cellular constructs are then subjected to suspension culture for several weeks. Traditional hESC differentiation techniques have yielded non-homogeneous EBs derived in standard static cultures providing an inefficient platform for cellular viability and embryonic modeling. Here, our study aimed at systematically comparing the formation, growth, and differentiation capabilities of hESC-derived hEBs in dynamic and static suspension cultures. We used a continuous flow perfusion slow turning lateral vessel, STLV, system (Synthecon) to model after an in vivo environment. This study is in part of a larger study investigating the role of HOXB5 in the human endothelial differentiation pathway. Embryoid bodies were created by hanging drops and then subjected to static or dynamic culture for 10 days. Cells were harvested and a simple Alkaline Phosphatase assay was used to determine if the system was viable for propagating hEB. We show that the STLV system is viable for our future studies and this system more efficient at maintaining hEBs.

European legal regulation of patents in the area of science and research Bioethics is an important part of law regulation in the medical field. According to the current state, bioethics is able to highlighted main issues, which are connected with medical research and suggest possible solution.This paper combines two controversial topics. First one is human embryonic stem cell research and second one is research on nanoparts and indicates Intelectual Property Law possibilities in this field. Paper is divided into two parts. First one deals with the legal regulation on research on embryo in the Czech Republic and in other states of The Western Europe. Main focus is based on patentability of research concerned with the human embryonic stem cells, which might have a great therapeutic potential but their preparation necessarily leads to the destruction of "human embryos". (HESC) Main concern is connected with regard to the European law and the current ground- breaking judgement, Brüstle v. Greenpeace eV. In mentioned judgment European Court of Justice held that after interpretation of the Directive on the legal protection of biotechnological inventions , it will not be able to grant a patent on research which led in the destruction of a human embryo. Paper also includes assessment of the attitude of the...

The area of stem cell research is rapidly evolving. One of the recent achievements is the capture of naïve human embryonic stem cells through reprogramming of primed human embryonic stem cells. In this thesis, Gene Co-expression Networks are used to further our understanding of naïve and primed human embryonic (hESCs). We found that GCNs of naïve and primed hESCs exhibits distinct network topological structures. Rewiring analysis of naïve and primed hESC GCNs showed significant rewiring and change in networks structures and behaviors. This demonstrates that naïve and primed hESCs are distinct cellular states. In addition, KLF genes circuitry, NANOG, and SOX2 were more active in naïve GCNs and formed more edges with other genes. Those genes were significantly rewired in our GCNs. We found that KLF5 is major player in our naïve hESCs GCNs. In addition, NANOG and SOX2 interacted only in naïve hESCs GCNs..The observations in our GCNs concerning KLF circuitry activity and, NANOG and SOX2 interactions were consistent with published stem cell literature. This demonstrates the power of GCNs in unfolding cellular characteristics and understanding the underlying gene dynamics.

A Dnmt3b é a principal DNA metiltransferase no processo de remetilação de regiões de DNA repetitivo do genoma durante a onda de reprogramação epigenética que ocorre no desenvolvimento embrionário. Sua expressão é regulada por uma série de mecanismos, entre os quais encontram-se os microRNAs (miRNAs). O objetivo deste trabalho foi investigar o papel dos mRNAs hsa-miR-203, hsa-miR-26a e hsa-miR-26b na regulação da expressão de DNMT3B durante a diferenciação de células-tronco embrionárias humanas e suas consequências para a metilação do DNA destas células. Para isto, geramos curvas de expressão dos miRNAs investigados, bem como da DNMT3B, a fim de verificar a existência de correlação entre elas. Também analisamos o efeito da superexpressão de miRNAs miméticos sobre a expressão de DMMT3B e o efeito da inibição da interação entre os miRNAs e a região 3\'UTR de DNMT3B sobre a expressão da DNA metiltransferase e sobre os níveis de metilação do DNA das células. Constatamos que a expressão de DNMT3B está inversamente correlacionada com a expressão dos miRNAs hsa-miR-203, hsa-miR-26a e hsa-miR-26b, mas não do hsa-miR-29b, regulador já validado para o controle da expressão de DNMT3B em células tumorais. Verificamos também que o impedimento da ligação do hsa-miR-203 ao segundo sítio predito para a ligação deste miRNA ao mRNA de DNMT3B ocasionou um aumento significativo na expressão da DNA metiltransferase, embora tal aumento não tenha refletido alteração nos níveis de metilação do DNA das células. Desta forma, concluímos que os miRNAs investigados fazem parte da maquinaria de regulação da DNA metiltransferase 3B na diferenciação de células-tronco embrionárias, embora o exato papel funcional de cada um deles no controle da expressão de DNMT3B e na metilação do DNA destas células ainda necessite ser melhor esclarecido / Dnmt3b is the major DNA methyltransferase in the proccess of repetitive DNA remethylation during the epigenetic reprogramming wave that occurs in the embryonic development. Its expression is regulated through several mechanisms, including microRNA (miRNAs). The aim of this study was to investigate the role of the miRNAs hsa-miR-203, hsa-miR-26a, and hsa-miR-26b in the regulation of DNMT3B expression during the differentiation of human embryonic stem-cells, and its consequences to the DNA methylation of these cells. To accomplish this, we generated expression curves for the investigated miRNAs, as well as for DNMT3B, to verify the existence of correlation between them. We also analysed the effect of superexpressing mimetic miRNAs on DNMT3B expression, and the effect of inhibiting the interaction of these miRNAs with DNMT3B 3\'UTR region on the DNA methyltransferase expression and the levels of DNA methyation. We found that DNMT3B expression is inversely correlated to hsa-miR-203, hsa-miR-26a, and hsa-miR-26b expression, but not to hsa-miR-29b, a previously validated DNMT3B regulator in tumor cells. We also verified that blocking the interaction of hsa-miR-203 to its predicted second interaction site at the DNMT3B mRNA significantly increased DNMT3B expression, but did not interfere with the levels of DNA methylation in these cells. Thus, we conclude that the investigated miRNAs are part of the DNMT3B regulatory machinery during the differentiation of embryonic stem-cells, although the exact functional role of each of them in the control of DNMT3B expression and in the DNA methylation of these cells still remains to be further explored

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Primary human hepatocytes are a scare resource with limited lifespan and variable function which diminishes with time in culture. As a consequence, their use in tissue modelling and therapy is restricted. Human embryonic stem cells (hESC) could provide a stable source of human tissue due to their self-renewal properties and their ability to give rise to all the cell types of the human body. Therefore, hESC have the potential to provide an unlimited supply of hepatocytes. To date, the use of hESCs-derived somatic cells is limited due to the undefined, variable and xeno-containing microenvironment that influences the cell performance and life span, limiting scale-up and downstream application. Therefore, the development of highly defined cell based systems is required if the true potential of stem cell derived hepatocytes is to be realised. In order to replace the use of animal derived culture substrates to differentiate and maintain hESCs-derived hepatocytes, an interdisciplinary approach was employed to define synthetic materials, which maintain hepatocyte-like cell phenotype in culture. A simple polyurethane, PU134, was identified which improved hepatocyte performance and stability when compared to biological matrices. Moreover, the synthetic polymer was amenable to scale up and demonstrated batch-to-batch consistency. I subsequently used the synthetic polymer surface to probe the underlying biology, identifying key modulators of hepatocyte-like cell phenotype. This resulted in the identification of a novel genetic signature, MMP13, CTNND2 and THBS2, which was associated with stable hepatocyte performance. Importantly, those findings could be translated to two hESC lines derived at GMP. In conclusion, hepatocyte differentiation of pluripotent stem cells requires a defined microenvironment. The novel gene signature identified in this study represents an example of how to deliver stable hESCs-derived hepatocytes.