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Item Axonal Outgrowth and Pathfinding of Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells(2020-08) Fligor, Clarisse; Meyer, Jason; Marrs, James; Belecky-Adams, Teri; Jones, Kathryn; Baucum, AJRetinal ganglion cells (RGCs) serve as a vital connection between the eye and the brain with damage to their axons resulting in loss of vision and/or blindness. Reti- nal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, providing a valuable model of RGC development in vitro. The working hypothesis of these studies is that hPSC-derived RGCs are capable of extensive outgrowth and display target specificity and pathfinding abilities. Initial efforts focused on charac- terizing RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner express- ing a compliment of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful to investigate and model the extensive axonal outgrowth necessary to reach post-synaptic targets. As such, additional efforts aimed to elucidate factors promoting axonal outgrowth. Results demonstrated significant enhancement of axonal outgrowth through modulation of both substrate composi- tion and growth factor signaling. Furthermore, RGCs possessed guidance receptors that are essential in influencing outgrowth and pathfinding. Subsequently, to de- termine target specificity, aggregates of hPSC-derived RGCs were co-cultured with explants of mouse lateral geniculate nucleus (LGN), the primary post-synaptic target of RGCs. Axonal outgrowth was enhanced in the presence of LGN, and RGCs dis- played recognition of appropriate targets, with the longest neurites projecting towards LGN explants compared to control explants or RGCs grown alone. Generated from xvii the fusion of regionally-patterned organoids, assembloids model projections between distinct regions of the nervous system. Therefore, final efforts of these studies focused upon the generation of retinocortical assembloids in order to model the long-distance outgrowth characteristic of RGCs. RGCs displayed extensive axonal outgrowth into cortical organoids, with the ability to respond to environmental cues. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC develop- ment, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing outgrowth as well as modeling long-distance projections and pathfinding abilities.Item Imaging and quantifying ganglion cells and other transparent neurons in the living human retina(National Academy of Sciences, 2017-11-28) Liu, Zhuolin; Kurokawa, Kazuhiro; Zhang, Furu; Lee, John J.; Miller, Donald T.; Engineering Technology, School of Engineering and TechnologyGanglion cells are the primary building block of retinal neural circuitry, but have been elusive to observe and quantify in the living human eye. Here, we show a light microscopy modality that reveals not only the somas of these cells, but also their 3D packing geometry, primary subtypes, and spatial projection to other neurons. The method provides a glimpse of the rich tapestry of neurons, glia, and blood vessels that compose the retina, thus exposing the anatomical substrate for neural processing of visual information. Clinically, high-resolution images of retinal neurons in living eyes hold promise for improved diagnosis and assessing treatment of ganglion cell and other neuron loss in retinal disease., Ganglion cells (GCs) are fundamental to retinal neural circuitry, processing photoreceptor signals for transmission to the brain via their axons. However, much remains unknown about their role in vision and their vulnerability to disease leading to blindness. A major bottleneck has been our inability to observe GCs and their degeneration in the living human eye. Despite two decades of development of optical technologies to image cells in the living human retina, GCs remain elusive due to their high optical translucency. Failure of conventional imaging—using predominately singly scattered light—to reveal GCs has led to a focus on multiply-scattered, fluorescence, two-photon, and phase imaging techniques to enhance GC contrast. Here, we show that singly scattered light actually carries substantial information that reveals GC somas, axons, and other retinal neurons and permits their quantitative analysis. We perform morphometry on GC layer somas, including projection of GCs onto photoreceptors and identification of the primary GC subtypes, even beneath nerve fibers. We obtained singly scattered images by: (i) marrying adaptive optics to optical coherence tomography to avoid optical blurring of the eye; (ii) performing 3D subcellular image registration to avoid motion blur; and (iii) using organelle motility inside somas as an intrinsic contrast agent. Moreover, through-focus imaging offers the potential to spatially map individual GCs to underlying amacrine, bipolar, horizontal, photoreceptor, and retinal pigment epithelium cells, thus exposing the anatomical substrate for neural processing of visual information. This imaging modality is also a tool for improving clinical diagnosis and assessing treatment of retinal disease.Item The Maturation of Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells and Their Degeneration in Glaucoma(2020-05) VanderWall, Kirstin B.; Androphy, Elliot; Cummins, Theodore R.; Berbari, Nicolas; Linnemann, Amelia; Meyer, Jason S.In glaucoma, the connection between the eye and the brain is severed leading to the degeneration of retinal ganglion cells (RGCs) and eventual blindness. A need exists to better understand the maturation of human RGCs as well as their degeneration, with the goal of developing new therapeutics diseases like glaucoma. Human pluripotent stem cells (hPSCs) provide an advantageous model for the study of RGC development and disease as they can be differentiated into RGCs in large, reproducible quantities. Efforts of the current studies initially focused on the development and maturation of RGCs from hPSCs. RGCs derived from hPSCs were a diverse population of cells and matured in a temporal fashion, yielding morphological and functional characteristics similar to their in vivo counterpart. CRISPR/Cas9 gene editing was then utilized to insert the OPTN(E50K) glaucomatous mutation into hPSCs to model RGC degeneration. RGCs harboring this mutation exhibited numerous degenerative phenotypes including neurite retraction an autophagy dysfunction. Within the retina, many cell types contribute to the health and maturation of RGCs including astrocytes. As such, a co-culture system of hPSC-derived RGCs and astrocytes was developed to better understand the interaction between these two cell types. When grown in co-culture with astrocytes, hPSC-derived RGCs demonstrated significantly enhanced and accelerated morphological and functional maturation, indicating an important relationship between these cells in a healthy state. Astrocytes have also been shown to encompass neurodegenerative phenotypes in other diseases of the CNS, with these deficits profoundly effecting the health of surrounding neurons. hPSC-derived astrocytes grown from OPTN(E50K)-hPSCs demonstrated cell autonomous deficits and exhibited significant effects on the degeneration of RGCs. Taken together, results of this study demonstrated the utilization of hPSCs to model RGC maturation and degeneration in glaucoma. More so, these results are one of the first to characterize astrocyte deficits caused by the OPTN(E50K) mutation and could provide a new therapeutic target for pharmacological screenings and cell replacement therapies to reverse blindness in optic neuropathies.