Evaluation and comparison of natural and synthetic retinoids on model of neural development

Abstract

Vitamin A and its derivatives, collectively termed retinoids, are a group of natural and synthetic molecules that are structurally and/or functionally analogous. Retinoids are essential for many biologically important processes during mammalian embryogenesis and adult homeostasis. Consequently, retinoids are often used to modulate cell proliferation and differentiation of cultured stem and progenitor cells in the laboratory, and possess the potential for use in numerous clinical applications. For example, retinoids are already applied as treatments of dermatological disorders, and as part of chemotherapeutic programmes for certain types of cancer. The importance of optimal retinoid function in embryonic neural development is well known, and is now being realised in certain adult neural progenitor cell populations. It has been shown that all-trans-retinoic acid CATRA), which activates all retinoic acid receptor subtypes, is the main active natural retinoid, inducing neural differentiation in several cell model systems, including embryonic stem cells and adult neural progenitors. The in vitro study of ATRA, however, is complicated by its photo-isomerisation and degradation when used under standard laboratory conditions. This instability has obvious disadvantages, for example, in stem cell biology where the ability to control the development of cells and tissues in a predictable way is paramount. Human stem cells present the opportunity to produce tissues that can be used for basic research into health and disease, development of pharmaceuticals, toxicological tests and ultimately cell replacement therapy. Biologists require molecules that induce reliable and reproducible biological activity resulting in consistent modes of cell differentiation. To address this issue, a small library of synthetic retinoids have been designed and prepared and their biological activity investigated. This thesis focused on the synthesised ATRA retinoid analogues, EC 19 and EC23 and aimed to evaluate their biological activity in relation to ATRA on a number of different embryonic and adult model systems. Unlike ATRA, these compounds do not isomerise in response to light or heat. Data show that both compounds are effective at inducing differentiation. In moc!el embryonic stem and adult progenitor cell culture systems both neural and non-neural cell sub-types are induced after EC 19 or EC23 treatment. Specifically, EC23 elicited similar cellular responses to ATRA when tested in vitro, inducing significant neural differentiation in the model cell 'systems and appears to do so via specific retinoic acid functional pathways. EC 19, however, induced different proportions of the cell-types observed after ATRA and EC23 media supplementation, inducing differentiation towards predominantly non-neural, epithelial-like phenotypes in stem cell models, and enhanced glial differentiation in adult progenitor models. Interestingly, EC19 also appeared to enhance the proliferation of a neuroblastoma model, in stark contrast to both ATRA and BC23 which induced a degree of neural differentiation. Both synthetic retinoids induce an up-regulation of cellular proteins involved with retinoic acid metabolism and signalling, suggesting that they elicit their effects directly via these pathways. Therefore EC 19 and EC23 represent promising candidates as alternatives to A TRA for directing cell differentiation and investigating the molecular pathways involved in neural cell development and retinoid modes of action in vitro. It is anticipated that this work will enhance research into retinoid mediated neuraldifferentiation. Finally, stable, synthetic modulators of cell-differentiation offer distinct advantages over existing technology and will be of significant commercial value.