Towards Retinal Repair: Bioelectric Assessment of Retinal Pigment Epithelium in vitro and Electrode Materials for Retinal Implants

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

Retinal Stem Cell Culture On Planar Scaffold For Transplantation In Animal Models Of Retinal Degeneration

Successful differentiation of pluripotent stem cells into retinal cells and subsequent transplantation in animal models have made stem cell-based therapy a closer possibility for treating retinal degenerations. However, major limitations are low efficiency in deriving desired cell types and continued need for large animal studies to validate small animal studies. In this study, we modified a biopolymer scaffold with laminin-521 as a novel substrate to culture retinal progenitor cells (RPCs). We hypothesize that laminin-521 will increase cell retention and provide a developmental signal for RPCs to mature and self-organize. The RPC scaffold culture was transplanted into a RD10 mice, a rodent model of retinal degeneration to evaluate the effects on disease pathology, and into pig eyes for a large animal pilot transplantation study. Differentiation of RPC was monitored by qRT-PCR, immunofluorescence and immunoblotting. Graft survival, integration, and host immune reactivity was assessed via histology and immunofluorescence. The laminin-521 coated scaffold (GCH-L521) increased cell attachment compared to non-coated scaffold. GCH-L521 was able to support growth and continued differentiation of RPC. When transplanted into animal models, the RPC scaffold graft survived in the host and did not cause significant host immune reaction. In RD10 mice, transplantation resulted in improved photoreceptor preservation. GCH-L521 can serve as a new substrate to culture RPC and simultaneously functions as a transplantation vehicle that is well-tolerated in the host

Mutation-independent treatment of autosomal dominant Retinitis Pigmentosa (adRP)

Viral-mediated gene therapy holds great promise for the treatment of severe inherited retinal diseases, such as Retintitis Pigmentosa (RP), which is caused by mutations in genes preferentially expressed in photoreceptor cells. The availability of vectors derived from the small adeno-associated virus (AAV) which efficiently and stably transduce the retina of animal models after intraocular administration strongly support the possibility to develop novel strategies for the treatment of such severe retinal degenerations otherwise incurable thus far. The main goals of my PhD project were: - generate artificial transcription repressors (ZFPs) targeted to the human rhodopsin promoter to silence at the transcriptional level the rhodopsin gene; - assess the efficacy of the treatment and the impact on the disease progression in the RP mouse model. Retinitis pigmentosa is by far the most studied inherited retinal disease. It is clinically and genetically heterogeneous recognizing autosomal recessive (arRP), autosomal dominant (adRP), X-linked, and digenic patterns of inheritance. More than 30 diseases genes have been identified so far and 12 of these have been associated with (adRP), representing between 15% and 35% of all cases. Despite recent success of the gene-based complementation approach for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc finger protein (ZFP)-based-repression of transcription may represent a novel and alternative mutation independent therapeutic approach for treating gain-of-function mutations, but proof-of-concept of this use is still lacking. In my PhD project we used a novel strategy to treat adRP based on zinc-finger-based artificial transcription factors (ZF-ATFs). These molecules can be engineered to silence genes carrying gain-of-function mutations that cause toxic effects into the cell where they are expressed. We generated ten artificial transcriptional repressors targeted to the human Rhodopsin which is the gene most commonly associated with adRP (20–30% of cases) with more than 150 mutations identified throughout its sequence, representing the most commonly mutated gene in RP. We characterized in vitro the ability of artificial transcriptional repressors to bind specifically the human rhodopsin promoter in order to exert a specific transcriptional control and we selected two out of ten functional zinc-finger-based repressors of rhodopsin. One of this was selected as the most efficient and was enclosed in an AAV2/8 for in vivo experiments. We demonstrated that the selected artificial zinc-finger-based repressors (ZFRs) resulted in a robust transcriptional repression of hRHO impacting disease progression in a mouse model of adRP over-expressing the P347S mutation. The data obtained support the use of ZFP-mediated silencing as a potentially relevant therapeutic strategy to treat gain of function mutations

Decellularised extracellular matrix-derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids

Tissue specific extracellular matrices (ECM) provide structural support and enable access to molecular signals and metabolites, which are essential for directing stem cell renewal and differentiation. To mimic this phenomenon in vitro, tissue decellularisation approaches have been developed, resulting in the generation of natural ECM scaffolds that have comparable physical and biochemical properties of the natural tissues and are currently gaining traction in tissue engineering and regenerative therapies due to the ease of standardised production, and constant availability. In this manuscript we report the successful generation of decellularised ECM-derived peptides from neural retina (decel NR) and retinal pigment epithelium (decel RPE), and their impact on differentiation of human pluripotent stem cells (hPSCs) to retinal organoids. We show that culture media supplementation with decel RPE and RPE-conditioned media (CM RPE) significantly increases the generation of rod photoreceptors, whilst addition of decel NR and decel RPE significantly enhances ribbon synapse marker expression and the light responsiveness of retinal organoids. Photoreceptor maturation, formation of correct synapses between retinal cells and recording of robust light responses from hPSC-derived retinal organoids remain unresolved challenges for the field of regenerative medicine. Enhanced rod photoreceptor differentiation, synaptogenesis and light response in response to addition of decellularised matrices from RPE and neural retina as shown herein provide a novel and substantial advance in generation of retinal organoids for drug screening, tissue engineering and regenerative medicine

Stem Cell Therapy: a Novel Approach for Vision Restoration in Retinitis Pigmentosa

Unfortunately, at present, degenerative retinal diseases such as retinitis pigmentosa remains untreatable. Patients with these conditions suffer progressive visual decline resulting from continuing loss of photoreceptor cells and outer nuclear layers. However, stem cell therapy is a promising approach to restore visual function in eyes with degenerative retinal diseases such as retinitis pigmentosa. Animal studies have established that pluripotent stem cells when placed in the mouse retinitis pigmentosa models have the potential not only to survive, but also to differentiate, organize into and function as photoreceptor cells. Furthermore, there is early evidence that these transplanted cells provide improved visual function. These groundbreaking studies provide proof of concept that stem cell therapy is a viable method of visual rehabilitation among eyes with retinitis pigmentosa. Further studies are required to optimize these techniques in human application. This review focuses on stem cell therapy as a new approach for vision restitution in retinitis pigmentosa

Stem Cell Therapy: a Novel Approach for Vision Restoration in Retinitis Pigmentosa

Unfortunately, at present, degenerative retinal diseases such as retinitis pigmentosa remains untreatable. Patients with these conditions suffer progressive visual decline resulting from continuing loss of photoreceptor cells and outer nuclear layers. However, stem cell therapy is a promising approach to restore visual function in eyes with degenerative retinal diseases such as retinitis pigmentosa. Animal studies have established that pluripotent stem cells when placed in the mouse retinitis pigmentosa models have the potential not only to survive, but also to differentiate, organize into and function as photoreceptor cells. Furthermore, there is early evidence that these transplanted cells provide improved visual function. These groundbreaking studies provide proof of concept that stem cell therapy is a viable method of visual rehabilitation among eyes with retinitis pigmentosa. Further studies are required to optimize these techniques in human application. This review focuses on stem cell therapy as a new approach for vision restitution in retinitis pigmentosa

Purinergic Signaling in the Retina: From Development to Disease

Retinal injuries and diseases are major causes of human disability involving vision impairment by the progressive and permanent loss of retinal neurons. During development, assembly of this tissue entails a successive and overlapping, signal-regulated engagement of complex events that include proliferation of progenitors, neurogenesis, cell death, neurochemical differentiation and synaptogenesis. During retinal damage, several of these events are re-activated with both protective and detrimental consequences. Purines and pyrimidines, along with their metabolites are emerging as important molecules regulating both retinal development and the tissue\u27s responses to damage. The present review provides an overview of the purinergic signaling in the developing and injured retina. Recent findings on the presence of vesicular and channel-mediated ATP release by retinal and retinal pigment epithelial cells, adenosine synthesis and release, expression of receptors and intracellular signaling pathways activated by purinergic signaling in retinal cells are reported. The pathways by which purinergic receptors modulate retinal cell proliferation, migration and death of retinal cells during development and injury are summarized. The contribution of nucleotides to the self-repair of the injured zebrafish retina is also discussed. © 2018 Elsevier Inc