The research in this thesis focuses primarily on two critical challenges that inhibit the late stage translation of cell-based therapies and Regenerative Medicines (RMs). These include product comparability after a change in manufacturing process or site; and the reimbursement of RMs, in particular those which target multiple simultaneous indications, or Multimorbidity . The automation and standardisation of stem cell cultures also represent key themes of this thesis, which may facilitate the development of scalable, reproducible manufacturing processes for cell-based therapies. Furthermore, given the current uncertainty regarding the characterisation and potency of Human Mesenchymal Stromal (or Stem) Cells (hMSCs) that has inhibited the successful clinical translation of hMSC-based products, understanding the characterisation and putative modes of action of these cells was also a priority throughout this research. Also, due to the increasing number of Human Embryonic Stem Cell (hESC) derived therapies progressing towards market, and the industry-wide shift towards Human Induced Pluripotent Stem Cells (hiPSC) as an alternative to hESCs, the measurement of the growth and characterisation of these cells types represents an important method of demonstrating product comparability after alternative manufacturing process steps in the present thesis. Finally, due to the potential of hiPSCs as a source of large numbers of hMSCs, the culture conditions required to direct the differentiation of hiPSCs to hMSCs are explored.

Neural crest induction and migration is the culmination of an intricate network of signalling from both the ectoderm and underlying mesoderm. Due to the transient nature of the neural crest, little is known about the specific interactions of growth factors and morphogens required for correct patterning. Neural crest stem cells were differentiated from embryonic stem cells in serum-free feeder-free culture conditions. Cells were further differentiated into functional peripheral neurons, expressing the synaptic protein neurexin3, able to spontaneously generate action potentials and showing responses to the neuroactive compounds GABA and NMDA. Microarray analyses and subsequent RT-qPCR experiments revealed that gene expression of the key pluripotency associated factor Oct-4 was initially downregulated upon differentiation before expression levels increased as cells developed into a neural crest phenotype. Further experiments indicated an intrinsic role for bone morphogenetic protein-4 (BMP-4) in mediating this resurgence of expression. In addition to BMP-4 influenced expression of Nanog, members of the developmental pluripotency associated family of genes and important adhesion molecules with roles in the epithelial to mesenchymal transition of the neural crest. Global gene expression profiling of differentiating functional neurons offers new insights into BMP-4 mediated patterning of the neural crest and peripheral nervous system during development. This study implicates BMP-4 as a key factor in neural crest differentiation, with a role in diverse cellular processes including proliferation, fate determination and cell migration. The role of BMP-4 in mediating expression of pluripotency and adhesion factors highlights a potential role in oncogenesis; neural crest cells share many phenotypic traits with cancer cells. Finally, the generation of functional peripheral neurons in feeder free culture conditions offers a reliable method for the recapitulation of these tissues and the possibility of use in future tissue replacement therapy.

LIN28 is an RNA-binding protein and a key regulator of developmental timing. It has dual functionality; preventing the maturation of microRNAs such as the let-7 family and increasing the translation efficiency of targeted mRNAs. LIN28 is highly expressed in human embryonic stem (hES) cells and is rapidly down regulated during neural maturation. Nuclear factor kappa B (NFkB) is a family of transcription factors most notable its role in inflammation. In 2009, it was reported that NFkB mediated cell transformation by directly upregulating UN28 transcription. However, in recent years a number of studies have demonstrated that NFkB plays an integral part in neurogenesis, when UN28 is transcriptionally silent. Activation of the canonical NFkB signalling pathway has been shown to be necessary for early neural stem cell (NSC) differentiation and inhibition of NFkB activity in vitro blocks neural maturation, although the mechanism remains elusive. The aim of this project is to clarify the roles of LIN28 and NFkB throughout neural specification of human embryonic stem (hES) cells in vitro using a reproducible targeted differentiation protocol. I have utilised lentiviral technology to modulate and monitor LIN28 and NFkB activity, respectively. Transcriptomic microarray analysis at key stages of neural differentiation indicated an inverse relationship between NFkB signalling and UN28.1 found that as neural progenitors (NPCs) differentiate, they downregulate LIN28 and become dependent on oxidative phosphorylation (OXPHOS) as a means of generating ATP. Furthermore, over extended passage, NPCs acquired high levels of NFkB activity and accumulated reactive oxygen species (ROS). Furthermore, late passage NPCs exhibited slower cell cycling, increased levels of cell cycle inhibitor p21 and critically an increased propensity for terminal differentiation. Constitutive activation of NFkB in early passage NPCs resulted in increased OXPHOS and p21 expression, suggesting that NFkB regulates metabolic activity and cell cycle progression during neural specification of hES cells.

Background: Moral distress is a poorly recognised and understood phenomenon and little is known of the triggering factors in ICU end-of-life decisions. Prior research focused largely on nurses, less was known about doctors’ experiences, and moral distress in relatives was under-investigated. Aim: To identify the triggers and constraints generating moral distress in end-of-life decisions in ICU and the consequences of moral distress for the clinical staff and relatives involved. Methods: A qualitative narrative thematic analysis of in-depth interviews with 20 bereaved relatives and 45 nurses and doctors closely involved in 21 patient cases of non-escalation and withdrawal of therapy, and organ donation following brain-stem death and circulatory death. The study was conducted in a large ICU in Northern Ireland (August 2012- November 2013) and funded by the Research and Development Office of the Public Health Agency. Key Findings: ICU staff and relatives experienced considerable moral distress in end-of-life care decisions. Bedside nurses and junior medical trainees with perceived lower levels of knowledge, experience and influence in end-of-life decisions experienced more moral distress than consultants and senior nurses. At least half of relatives experienced moral distress at some point along the ICU end-of-life care trajectory. Triggers specific to all three participant groups included breaches in the consistency and continuity of care delivery and end-of-life decisions and insensitive and lengthy organ retrieval procedures. In particular, failure to ensure ‘The Good Death’ left a powerful sense of failed obligation. Some relatives continued to experience the aftermath of moral distress several months after the death. Conclusions: Findings have important implications for (a) educational preparation of new ICU nurses and doctors to prepare them for the complexities of the ethically challenging ICU environment: (b) the support of relatives in ICU with follow-up after the death; and (c) improvement in organ retrieval services in Northern Ireland.

Mesenchymal stromal cells (MSCs) reside within the perivascular niche and have been shown in vivo to facilitate vascular repair. Previous in vitro studies, have demonstrated the potential of MSCs to differentiate towards an endothelial lineage, when cultured at high cell density; but the characterisation of these cells and the mechanisms directing this important differentiation effect are ill-defined. To resemble a three-dimensional (3D) cellular environment, MSCs were cultured as spheroids and the endothelial characteristics of these cells determined. MSCs cultured as spheroids significantly increased their expression of the endothelial markers; PECAM-1, Tie2, VE-cadherin and vWF, when compared to MSCs cultured in close cell contact as a two-dimensional (2D) monolayer. In addition, MSCs cultured as 3D spheroids behaved as functional endothelial cells in vitro; including the ability to uptake low-density lipoproteins, secretion of nitric oxide and the ability to form network-like structures. MSC spheroids exhibited significantly increased levels of Notch signalling, compared to 2D MSCs in close cell contact, which caused a significant decrease in the endothelial characteristics when inhibited. Conversely, activation of Notch signalling caused a significant and specific increase in the expression of PECAM-1, which was regulated by the Notch ligands Jagged1 and DLL4. Thus, Notch signalling is a crucial pathway that controls PECAM-1 expression and regulates the angiogenic fate of MSCs within spheroids. This study has therefore identified an efficient culture model and key signalling mechanism which may be used to induce MSCs towards an angiogenic fate for vascular repair and regeneration therapies.

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The oncoprotein MYC has long been recognized as an important stem cell regulator, yet its direct biological contributions have been difficult to determine. MYC activation can induce pleiotropic phenotypes and mediates cellular functions as opposing as cell growth and proliferation, metabolism, differentiation and apoptosis. In addition, functional redundancy with MYCL and MYCN proteins as well as dose dependency, complicates the identification of the most relevant biological functions. Studies in tissues with high proliferative capacity and rapid turnover have shown that MYC is a key regulator of homeostasis by balancing stem cell self-renewal, proliferation and differentiation processes. In skin, MYC induces the exit of epidermal stem cells from their niche, increases proliferation of progenitor cells and subsequently stimulates lineage specific differentiation into interfollicular epidermis and sebaceous glands; yet the direct transcriptional roles of MYC in these processes remained elusive. To gain insight into the transcriptional roles of MYC in epidermal stem cell homeostasis, I performed chromatin immunoprecipitation on microarrays (ChIP-on-Chip) using mouse proximal promoter arrays combined with mRNA expression data that was generated using epidermal cells from wild-type and transgenic K14MycER mice, treated in a time-course from zero to six days with tamoxifen, to induce the Myc transgene expression in the basal undifferentiated layers of the epidermis. Data analysis revealed that 2187 genes, which corresponds to 15% of the promoter regions covered, were directly regulated by MYC. To identify genes uniquely regulated by MYC in skin, I performed gene expression studies on mouse skin in which MYC was conditionally deleted in the basal layer of the epidermis. Remarkably, I found that 45% of all repressed genes were related to epidermal maintenance and differentiation. To better understand the mechanism of how MYC induces keratinocytes to differentiate specifically into lineages of sebaceous glands and interfollicular epidermis, I analyzed whether MYC might have directly regulated genes involved in skin differentiation. Here, I focused my studies on a single 2.2 Mb locus located on mouse chromosome 3 designated as the epidermal differentiation complex (EDC). To assess how activation of MYC could influence the expression of genes localized to the EDC, I performed ChIP-on-Chip for MYC, H3K4me3, H3K27me3, as well as transcription factors, which have been described to regulate terminal differentiation in skin, such as CEBPα, OVOL-1, KLF4, TCFAP2-γ and SIN3A, among others. I demonstrated that MYC recruits a specific set of tissue-specific transcription factors to the EDC, (e.g. KLF4 and OVOL-1) and thereby prevents binding of a different and distinct set of genomic regulators, (e.g. CEBPα , MXI1 and SIN3A). Using a combination of mouse models and systems biology tools, I then identified SIN3A as a key regulator in this MYC-dependent transcriptional network. I found that MYC and SIN3A form a negative feedback loop, which is required to balance proliferation and differentiation in epidermis, and both factors are essential to maintain skin homeostasis.

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