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.

Cataract surgery is currently the most common surgical procedure done in the world. However, within 5 years, approximately half of these patients will develop posterior capsule opacification (PCO). In cataract surgery, the biological lens is replaced with an intraocular lens (IOL). PCO is caused by migration and transformation of residual lens epithelial cells (LEC) that remain in the capsule following the surgery. LECs which have migrated to the posterior capsule within the first month of surgery are thought to be the major contributors to PCO since after this time, the capsule completely seals. A mathematical model has been developed in order to better understand the process of LEC migration during PCO. The model addresses the impact of substrate and substrate modification as well as the presence and absence of the growth factors transforming growth factor beta (TGF(beta)) and fibroblast growth factor (FGF2). It was developed from a first order rate of decay model taken from process control. If the cell speed is divided by the distance travelled by the cell up to the point of posterior capsule breach, the time for the LECs to breach the capsule posterior can be calculated. The model was tested with literature data and was able to predict the effects of cell speed on the presence of various extracellular matrix components and growth factors. It was determined that potentially modification with fibronectin may be useful for the prevention of PCO Preliminary experimental validation of the model was performed by modifying silicone substrates with various extracellular matrix derived peptides. Results demonstrate that peptide modified surfaces may be more resistant to EMT by increasing cell adhesion and decreasing cell migration. Therefore, this LEC migrational model will be a useful tool in the development of superior IOLs and materials. / Thesis / Master of Applied Science (MASc)

H αλόγιστη απόρριψη των αγροτοβιομηχανικών αποβλήτων στο περιβάλλον προκαλεί σοβαρά προβλήματα παγκοσμίως. Πολλά από αυτά όπως π.χ. τυρόγαλα περιέχουν θρεπτικά συστατικά τα οποία μπορούν να χρησιμοποιηθούν από μικροοργανισμούς προς παραγωγή προϊόντων υψηλής προστιθέμενης αξίας. Στην παρούσα εργασία χρησιμοποιήθηκε ο ζυμομύκητας Kluyveromyces marxianus για την ζύμωση συνθετικού μέσου με λακτόζη συγκέντρωσης ανάλογης με εκείνη του τυρογάλακτος (~5% w/v) προς παραγωγή αιθανόλης. Προσδιορίσθηκαν οι βέλτιστες φυσικοχημικές συνθήκες και τα αποτελέσματα έδειξαν ότι σε pH 7 και 30οC επιτυγχάνεται μέγιστη ταχύτητα ζύμωσης και συγκέντρωση αιθανόλης. Στις ίδιες συνθήκες, μέτρηση του ρυθμού πρόσληψης λακτόζης επισημασμένης με 14C από τον μικροοργανισμό έδειξε ότι αυτός σχετίζεται άμεσα με την κινητική της ζύμωσης. / Τhe indiscriminate disposal of agro waste in the environment causes serious problems worldwide. However, many of them for example whey, contain nutrients which can be used by microorganisms in order to produce products with high value. In this study is being used the yeast Kluyveromyces Marxianus for the fermentation of synthetic substrate of lactose with lactose concentration similar to that of the whey (~5% w/v) for ethanol production. Having determined the optimal physicochemical conditions and the results showed that at pH 7 and 30οC achieved a maximum speed of fermentation and ethanol concentration. Under the same conditions, measuring the rate of uptake of 14C-labeled lactose by the microorganism showed that this is directly related to the kinetics of fermentation