Human senses are specifically designed to recognize and understand the world that surrounds us. Even though we have five senses, vision alone is responsible for at least 30 % of the sensory input to our brain. The visual process is initiated in a highly specialized cell type, the photoreceptors. These are light-sensitive cells located in the retina, a layered nervous tissue situated at the back of the eye. Retinal degeneration diseases are a highly heterogeneous group of conditions that include mutations affecting the survival, maintenance and proper functioning of photoreceptors or the adjacent retinal pigment epithelium (RPE). Such mutations, alone or in combination with environmental factors, cause the loss of the affected cells, and therefore, impairment of the visual sense. Retinitis Pigmentosa and Age-related Macular Degeneration are typical examples of retinal degenerative diseases eventually leading to blindness. In the first one, rod photoreceptors degenerate and consequently also cone photoreceptors are lost. The second is characterized by malfunction and loss of both, RPE and photoreceptor cells. Many current therapeutic approaches for the treatment of retinal degenerative diseases focus on slowing down the progression of the disease, rather than restoring the visual function. Currently, new therapies with the potential to recover the visual signal are under development. Some of these therapeutic strategies have already reached clinical stages, including gene therapy or retinal prosthesis. However, gene therapy approaches require the presence of remaining photoreceptors and, furthermore, particular targeting of disease-related genes. Retinal prosthesis still require improvement in terms of long-term biocompatibility and relevant visual function recovery. An alternative strategy for vision restoration is cell replacement of the lost photoreceptors, which is potentially suitable for targeting late stages of retinal degeneration diseases, independently of the inherent cause of the disease. Human vision relies primarily on cone photoreceptors, which are the cells responsible for color and high acuity vision under daylight conditions. However, cones represent a minority of the photoreceptors within the retina, and so, due to the low availability of these cells, cone photoreceptor transplantation studies lag behind rod transplantation studies. Consequently, in this study, strategies to increase the numbers of cone photoreceptors within mouse embryonic stem cells (mESC)-derived retinal organoids, which represent a potential cell source for transplantation studies, were explored. In this regard, I manipulated developmental pathways known to be involved in retinal development, such as Notch signaling, through the addition of various compounds in the retinal organoid maturation media. However, early cone markers have not yet been definitively identified, complicating the detection and isolation of cone photoreceptor precursors within the organoids. Therefore, a new early cone-reporter mESC line was generated in the course of this study as a valuable tool with the potential to facilitate the development of novel cone photoreceptor replacement therapies. Equally important in the field of photoreceptor cell replacement is the understanding of how the transplanted donor cells interact with the host retina. Previous studies have shown that visual function improvement is possible after transplanting rod or cone-like photoreceptor precursors into the sub-retinal space of mouse models for retinal degeneration. For many years it has been assumed that the underlying mechanism for the observed vision improvement was the migration and structural integration of donor cells into the host outer nuclear layer, where they mature and establish synaptic connections with the host retinal circuitry. However, experiments performed in this study demonstrate, for the first time, that upon transplantation donor and host photoreceptors exchange cytoplasmic material rather than structurally integrate into the host outer nuclear layer. Furthermore, insights into the transferred cytoplasmic content are given, i.e. that mRNA, but not mitochondria are exchanged by donor and host photoreceptors. This novel way of photoreceptor-photoreceptor communication led to a paradigm change in the field of retinal transplantation, requiring a re-interpretation of former transplantation studies. In addition, the discovery of the material transfer phenomenon might serve as a starting point for the development of novel therapeutic strategies based on cell-cell support for the treatment of retinal degenerative diseases. This study generated new knowledge in two important topics related to the development of cell therapies for retinal degeneration diseases, including the development of tools for cone transplantation studies as well as elucidating the interaction between donor and host cells upon transplantation.

Indiana University-Purdue University at Indianapolis (IUPUI) / Human pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some in vitro approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different in vitro systems that could help better mimic certain physiological systems that tend to not be mimicked by in vivo models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction. Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in in vivo models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using in vivo models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.

<p>Human pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some <em>in vitro</em> approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different <em>in vitro</em> systems that could help better mimic certain physiological systems that tend to not be mimicked by <em>in vivo</em> models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction.  Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in <em>in vivo</em> models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using <em>in vivo</em> models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.  </p>