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The number of people affected by degenerative diseases of the retina increases notably due to the longer life expectancy. The weakening of the sight concerns especially older people, who suffer from degeneration of the retina and its retinal pigment epithelium (RPE) monolayer. Currently, these degenerative diseases of the retina are incurable. Cell transplantation of healthy RPE cells is considered to be a potential treatment for these diseases. Due to the unlimited self-renewal capacity and pluripotency human pluripotent stem cells (hPSCs) are considered as possible cell sources for transplantation purposes. However, the current differentiation protocols for RPE cells from hPSCs are inefficient and result in low yields. The aim of this thesis was to study whether biodegradable poly(L-lactide) (PLLA) microparticles and commercial biostable cellulose anion exchange diethylaminoethyl (DE) 53 microparticles could be exploited in increasing the efficacy of differentiation of hPSCs into RPE cells. In addition, the potential of the microparticle culture system for stem cell maintenance in feeder-free conditions was evaluated. The attachment and proliferation of hPSCs on microparticles were analyzed by immunofluorescence analysis and microscopy. The success of differentiation of RPE from stem cells in microparticle cultures was evaluated by inspecting the appearance of pigmentation in cultures as well as by investigating the expression of RPE maturation markers on gene expression levels by RT-PCR. The PLLA microparticles did not support the attachment and proliferation of hPSCs in feeder-free conditions. The poor performance of PLLA particles in cell culture studies might be due to the hydrophobic nature of the particle surfaces. In contrast, hPSCs attached, proliferated and maintained their pluripotency on DE 53 particles. Furthermore, the hPSCs were successfully differentiated into RPE cells on these DE 53 particles. However, the differentiation efficacy did not outperform the conventional differentiation protocol. This was the first study in which the hPSCs were successfully differentiated into RPE cells on microparticles. Microparticles are far from being routinely used in hPSC maintenance and differentiation to mature cell types. More long-term and large-scale studies need to be conducted in the future. Nevertheless, at the moment they seem appealing and promising with a possibility to grow stem cells in large-scale and cost effectively. The microparticle culture systems set challenges for the cell analysis due to their 3-dimensional structure and autofluorescence property. Therefore, novel methods for characterization, analysis and imaging of microparticle culture systems should be developed. /Kir12

The aim of this thesis was to develop methods for future solutions to prevent eye diseases caused by the dysfunctions of retinal pigment epithelial (RPE) cells and to restore the vision of blind patients. On a cellular level, the degeneration of RPE cells is often the prime cause of eye diseases such as age-related macular degeneration and some forms of retinitis pigmentosa. RPE cell replacement therapy may provide new solutions for the prevention of eye diseases that lead to blindness. RPE cells differentiated from pluripotent stem cells provide a promising source for cell replacement therapy. However, the functionality of the differentiated cells is still not fully proven. One objective of this thesis was to provide solutions for testing the functionality of differentiated RPE cells. If blindness cannot be cured, artificial vision provided by retinal implant may be considered. The second objective of this thesis was to characterize the electrochemical properties of the different electrode materials used in retinal implants. The electrode materials used in retinal implants should be carefully considered in order to increase the resolution of the implant and to provide stable, safe, and biocompatible charge injection. All the methods used and developed in this thesis were based on bioelectrical phenomena. The electrochemical characterization of five different electrode materials used in retinal implants used electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measurements. We considered the effect of electrode size and material on charge capacity and impedance. Atomic force microscopy (AFM) was used to study the surface properties of the studied electrodes. The testing of the materials was done using exactly the same measurement conditions and electrode producing methods to provide easily comparable data. In this thesis, the functionality of RPE cells differentiated from human embryonic stem cells (hESC-RPE) was studied with two different methods. EIS was used to compare the electrical properties between two different RPE cell lines (immortalized human RPE cell line (ARPE-19) and hESC-RPE). To our knowledge, EIS measurements of RPE cells have not been published before. EIS was also used to find out how the barrier properties of hESC-RPE cells differ when the cells are in different stages of maturity. In addition, we developed a method that could be used to study the functionality of hESC-RPE cells with in vitro electroretinography (ERG) measurements: Our hypothesis is that RPE cells enhance the ERG response of the mouse retina and enable longer culturing of the functional retina in vitro. Comparing the ERG responses of a mouse retina alone and of a mouse retina cultured together with hESC-RPE cells could reveal the functionality of hESC-RPE cells. The EIS measurements were in accordance with biological analyses. The hESC-RPE cells resembled morphologically mature RPE, and thus created high transepithelial resistance (TER) indicating high integrity and tight junction formation. The EIS measurements revealed that during the maturation the TER of the cell culture increases, peak phase diagram shifts to lower frequencies, and the capacitance of the epithelium increases. Permeability measurements verified that EIS measurements reveal the tight junction failures and integrity decrease caused by calcium chelation. With the developed setup we were able to measure ERG responses from both the co-culture of retina and RPE and the retina cultured alone. However, due to limited sample size and possibly due to short co-culture time in our culture setup as yet we were not able to prove the hypothesis by showing that RPE cells would enhance the ERG response of the retina in vitro. Both the retina cultured alone and the co-culture responded to light stimulus after one day of culturing. CV and EIS measurements of different electrodes showed that iridium-black (Ir-b) and platinum-black (Pt-b) electrodes were superior, i.e. they had higher charge injection capacity and lower impedance when compared to other tested materials (gold (Au), titaniumnitrate (TiN), titanium (Ti)). Based on our findings we can conclude that novel biocompatible electrode materials that have the potential to be used in implantation are available. In the same way as in this thesis, the electrochemical testing of electrode materials should be done using similar testing methods for every material to enable easy comparison of the results between different materials. At the moment, cell replacement therapy and the use of RPE cells is seriously considered as a choice for eye disease treatment. Our results suggest that EIS is useful when evaluating the overall maturity, integrity, and functionality of the RPE cell culture. In forthcoming cell transplantation therapies, EIS could provide a means to test the validity of stem cell-derived RPE non-invasively and aseptically before implantation. Our initial tests show that studies to test the ability of RPE cells to rescue the photoreceptors in a mouse model by testing ERG responses in vitro should be continued. Even though our results did not produce conclusive evidence, the co-culture of the retina and hESC-RPE cells may be a useful in vitro model for investigating the RPE cell replacement therapy and possible drug releasing materials for the retina.