Stem cell technologies as a branch of regenerative medicine are becoming increasingly popular as the science behind it evolves. Therefore, it is important that the regulatory framework pertaining to stem cell technologies be well defined and appropriate to prevent unethical and unscrupulous behaviour on the part of medical practitioners, which gives rise to stem cell tourism. South African legislation pertaining to stem cell technology is regarded as inadequate and dissonant with the Constitution, exacerbating the problem of stem cell tourism and denying patients access to certain stem cell therapies, which ultimately can be viewed as an infringement of their constitutional rights. The United Kingdom (UK) provides a clear-cut regulatory framework, which is not only centred around consent and patient safety but is also conducive to production of stem cell therapies. For such reasons, this dissertation finds the UK framework to be an appropriate benchmark against which the South African regulatory framework can be evaluated. By means of comparison and elaborating on the biology of stem cells in addition to pertinent ethical principles, legislation and human rights of both South Africa and the UK, an argument will be made out that South African legislation pertaining to stem cell therapy and related matters is wanting. Furthermore, analysis will be made of the definition of biological medicine as put forward by the Medicines and Related Substances Control Act 101 of 1965 to conclude that certain stem cell therapies are best excluded from such a definition as such stringent requirements and protocols encumbers access to stem cell therapies and inflates costs. Lastly, remedial measures are proposed to remedy these injustices by proposing for the institution of a specialist adivisary committee to oversee stem cell and related activities. Key Words: Regenerative Medicine; Stem Cells; Stem Cell Regulation; National Health Act; Medicines and Related Substances Control Act; Advanced Therapy Medicinal Product; Human Tissue Authority; Human Fertilisation and Embryology Authority; HTA; HFEA; Medicine and Healthcare Products Regulatory Agency; MHRA; European Medicines Agency; Tissue-engineered Products; Doctor-Patient Relationship; Medical Innovation Bill 2014; Experimental Treatments; Innovative Therapy; Hospital Exemption; Informed Consent; Special Exemption; Autologous Stem Cell Therapy; Stem Cell Transplants; Gene Therapy Advisory Committee. / Dissertation (LLM)--University of Pretoria, 2017. / Public Law / LLM / Unrestricted

<p>Mammalian development is orchestrated by global transcriptional changes, which drive cellular differentiation, giving rise to diverse cell types. The mechanisms that mediate the temporal control of early differentiation can be studied using embryonic stem cell (ESCs) and embryonal carcinoma cells (ECCs) as model systems. In these stem cells, differentiation signals induce transcriptional repression of genes that maintain pluripotency (PpG) and activation of genes required for lineage specification. Expression of PpGs is controlled by these genes’ proximal and distal regulatory elements, promoters and enhancers, respectively. Previously published work from our laboratory showed that during differentiation of ESCs, the repression of PpGs is accompanied by enhancer silencing mediated by the Lsd1/Mi2-NuRD-Dnmt3a complex. The enzymes in this complex catalyze histone H3K27Ac deacetylation and H3K4me1/2 demethylation followed by a gain of DNA methylation mediated by the DNA methyltransferase, Dnmt3a. The absence of these chromatin changes at PpG enhancers during ESC differentiation leads to their incomplete repression. In cancer, abnormal expression of PpG is commonly observed. Our studies show that in differentiating F9 embryonal carcinoma cells (F9 ECCs), PpG maintain substantial expression concomitant with an absence of Lsd1-mediated H3K4me1 demethylation at their respective enhancers. The continued presence of H3K4me1 blocks the downstream activity of Dnmt3a, leading to the absence of DNA methylation at these sites. The absence of Lsd1 activity at PpG enhancers establishes a “primed” chromatin state distinguished by the absence of DNA methylation and the presence of H3K4me1. We further established that the activity of Lsd1 in these cells was inhibited by Oct3/4, which was partially repressed post-differentiation. Our data reveal that sustained expression of the pioneer pluripotency factor Oct3/4 disrupts the enhancer silencing mechanism. This generates an aberrant “primed” enhancer state, which is susceptible to activation and supports tumorigenicity. </p> <p>As differentiation proceeds and multiple layers of cells are produced in the early embryo, the inner cells are depleted of O₂, which triggers endothelial cell differentiation. These cells form vascular structures that allow transport of O₂ and nutrients to cells. Using ESC differentiation to endothelial cells as a model system, studies covered in this thesis work elucidated a mechanism by which the transcription factor Vascular endothelial zinc finger 1 (Vezf1) regulates endothelial differentiation and formation of vascular structures. Our data show that Vezf1-deficient ESCs fail to upregulate the expression of pro-angiogenic genes in response to endothelial differentiation induction. This defect was shown to be the result of the elevated expression of the stemness factor Cbp/p300-interacting transactivator 2 (Cited2) at the onset of differentiation. The improper expression of Cited2 sequesters histone acetyltransferase p300 from depositing active histone modifications at the regulatory elements of angiogenesis-specific genes that, in turn, impedes their activation. </p> <p>Besides the discovery of epigenetic mechanisms that regulate gene expression during differentiation, our studies also include development of a sensitive method to identify activities of a specific DNA methyltransferase at genomic regions. In mammals, DNA methylation occurs at the C5 position of cytosine bases. The addition of this chemical modification is catalyzed by a family of enzymes called DNA methyltransferases (Dnmts). Current methodologies, which determine the distribution of Dnmts or DNA methylation levels in genomes, show the combined activity of multiple Dnmts at their target sites. To determine the activity of a particular Dnmt in response to an external stimulus, we developed a method, Transition State Covalent Crosslinking DNA Immunoprecipitation (TSCC-DIP), which traps catalytically active Dnmts at their transition state with the DNA substrate. Our goal is to produce a strategy that would enable the determination of the direct genomic targets of specific Dnmts, creating a valuable tool for studying the dynamic changes in DNA methylation in any biological process.</p>

Electrospun nanofibers have been researched extensively in the culturing of stem cells to understand their behavior since electrospun fibers mimic the native extracellular matrix (ECM) in many types of mammalian tissues. Here, electrospun nanofibers with defined structure (orientation/alignment) and pore size could significantly modulate human mesenchymal stem cell (hMSC) behavior. Controlling the fiber membrane pore size was predominantly influenced by the duration of electrospinning, while the alignment of the fiber membrane was determined by parallel electrode collector design. Electric field simulation data provided information on the electrostatic interactions in this electrospinning apparatus.hMSCs on small-sized pores (~3-10 µm²) tended to promote the cytoplasmic retention of Yes-associated protein (YAP), while larger pores (~30-45 µm²) promoted the nuclear activation of YAP. hMSCs also displayed architecture-mediated behavior, as the cells aligned along with the fiber membranes orientation. Additionally, fiber membranes affected nuclear size and shape, indicating changes in cytoskeletal tension, which coincided with YAP activity. The mechanistic understanding of hMSC behavior on defined nanofiber structures seeks to advance their translation into more clinical settings and increase biomanufacturing efficiencies.