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Item Astrocytes modulate neurodegenerative phenotypes associated with glaucoma in OPTN(E50K) human stem cell-derived retinal ganglion cells(Elsevier, 2022) Gomes, Cátia; VanderWall, Kirstin B.; Pan, Yanling; Lu, Xiaoyu; Lavekar, Sailee S.; Huang, Kang-Chieh; Fligor, Clarisse M.; Harkin, Jade; Zhang, Chi; Cummins, Theodore R.; Meyer, Jason S.; Medical and Molecular Genetics, School of MedicineAlthough the degeneration of retinal ganglion cells (RGCs) is a primary characteristic of glaucoma, astrocytes also contribute to their neurodegeneration in disease states. Although studies often explore cell-autonomous aspects of RGC neurodegeneration, a more comprehensive model of glaucoma should take into consideration interactions between astrocytes and RGCs. To explore this concept, RGCs and astrocytes were differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated OPTN(E50K) mutation along with corresponding isogenic controls. Initial results indicated significant changes in OPTN(E50K) astrocytes, including evidence of autophagy dysfunction. Subsequently, co-culture experiments demonstrated that OPTN(E50K) astrocytes led to neurodegenerative properties in otherwise healthy RGCs, while healthy astrocytes rescued some neurodegenerative features in OPTN(E50K) RGCs. These results are the first to identify disease phenotypes in OPTN(E50K) astrocytes, including how their modulation of RGCs is affected. Moreover, these results support the concept that astrocytes could offer a promising target for therapeutic intervention in glaucoma.Item Astrocytes Regulate the Development and Maturation of Retinal Ganglion Cells Derived from Human Pluripotent Stem Cells(Elsevier, 2019-02-12) VanderWall, Kirstin B.; Vij, Ridhima; Ohlemacher, Sarah K.; Sridhar, Akshayalakshmi; Fligor, Clarisse M.; Feder, Elyse M.; Edler, Michael C.; Baucum, Anthony J.; Cummins, Theodore R.; Meyer, Jason S.; Biology, School of ScienceRetinal ganglion cells (RGCs) form the connection between the eye and the brain, with this connectivity disrupted in numerous blinding disorders. Previous studies have demonstrated the ability to derive RGCs from human pluripotent stem cells (hPSCs); however, these cells exhibited some characteristics that indicated a limited state of maturation. Among the many factors known to influence RGC development in the retina, astrocytes are known to play a significant role in their functional maturation. Thus, efforts of the current study examined the functional maturation of hPSC-derived RGCs, including the ability of astrocytes to modulate this developmental timeline. Morphological and functional properties of RGCs were found to increase over time, with astrocytes significantly accelerating the functional maturation of hPSC-derived RGCs. The results of this study clearly demonstrate the functional and morphological maturation of RGCs in vitro, including the effects of astrocytes on the maturation of hPSC-derived RGCs.Item Elucidating Cellular Mechanisms Underlying Retinal Ganglion Cell Neurodegeneration in a Human Pluripotent Stem Cell-Derived Model(2022-12) Huang, Kang-Chieh; Cummins, Theodore R.; Meyer, Jason S.; Marrs, James A.; Perrin, Benjamin J.; Lasagna Reeves, Cristian A.Glaucoma is a leading cause of blindness characterized by the progressive loss of retinal ganglion cells (RGCs), essentially severing the connection between the eye and the brain. Among many underlying causes of the disease, mutations in the Optineurin (OPTN) gene result in severe RGC neurodegeneration in the absence of elevated intraocular pressure, providing a novel opportunity to study molecular mechanisms that lead to RGC neurodegeneration associated with glaucoma. Efforts of this study establishing a human pluripotent stem cell (hPSC)-derived in vitro disease model by inserting OPTN(E50K) mutation via CRISPR/Cas9 genome editing and investigate the cellular mechanisms of RGC neurodegeneration associated with glaucoma. OPTN(E50K) RGCs revealed neurodegeneration phenotypes, including downregulation of RGCs transcription factors, neurite retraction, and hyperexcitability, suggesting that OPTN(E50K) RGCs can serve as an appropriate disease model to study glaucoma-associated neurodegeneration. Since OPTN serves a primary role as an autophagy receptor, we further hypothesized that the OPTN(E50K) mutation disrupts autophagy in RGCs, and modulation of autophagy by mammalian target of rapamycin (mTOR)-independent pathways can preserve RGC phenotypes by maintaining mTOR signaling. OPTN(E50K) RGCs exhibited a higher number of OPTN puncta along with an overall reduced expression of OPTN protein, indicating a gain of toxic protein accumulation or loss of protein function. Furthermore, OPTN(E50K) RGCs revealed an accumulation of the autophagosome protein LC3 in a punctal manner as well as increased expression of lysosomal proteins, suggesting a disruption of degradation pathway in autophagosome and lysosome fusion. As mTOR complex 1 (mTORC1) signaling serves as a negative regulator of autophagy, a downregulation of mTORC1 signaling via activation of stress sensor adenosine monophosphate-activated protein kinase (AMPK) was observed as a possible compensatory mechanism for autophagy deficits in OPTN(E50K) RGCs. Pharmacological inhibition of mTOR in wild-type hRGCs resulted in similar disease-related phenotypes, while preservation of the mTOR pathway in OPTN(E50K) RGCs by treatment with the mTOR-independent autophagy modulator trehalose cleared OPTN accumulated puncta, preserving mTORC1 signaling, as well as rescuing neurodegenerative phenotypes. To further validate these associations in an animal model, the microbead occlusion mouse model was established by injection of magnetic microbeads in the anterior chamber to block aqueous outflow resulting ocular hypertension. In agreement with our findings in hRGCs, a decrease in mTOR signaling associated with an increase in the expression of autophagy-associated proteins was observed in RGCs in the microbead occlusion model. Additionally, these disease-related phenotypes were observed specifically within RGCs but not cortical neurons with an underlying OPTN(E50K) mutation, demonstrating that autophagy represents an essential pathway in RGCs to maintain homeostasis, and selective disrupt of autophagy in RGCs leads to neurodegeneration. Taken together, the results of this study highlight an essential balance between autophagy and mTORC1 signaling that is essential for the homeostasis of RGCs, while disruption to these signaling pathways contributes to neurodegenerative features in glaucoma. These results also demonstrated the ability to pharmacologically intervene to experimentally manipulate these pathways and rescue neurodegenerative phenotypes, providing a potential therapeutic target to prevent glaucoma-associated neurodegeneration.Item Extension of retinofugal projections in an assembled model of human pluripotent stem cell-derived organoids(Cell Press, 2021-09-14) Fligor, Clarisse M.; Lavekar, Sailee S.; Harkin, Jade; Shields, Priya K.; VanderWall, Kirstin B.; Huang, Kang-Chieh; Gomes, Cátia; Meyer, Jason S.; Biology, School of ScienceThe development of the visual system involves the coordination of spatial and temporal events to specify the organization of varied cell types, including the elongation of axons from retinal ganglion cells (RGCs) to post-synaptic targets in the brain. Retinal organoids recapitulate many features of retinal development, yet have lacked downstream targets into which RGC axons extend, limiting the ability to model projections of the human visual system. To address these issues, retinal organoids were generated and organized into an in vitro assembloid model of the visual system with cortical and thalamic organoids. RGCs responded to environmental cues and extended axons deep into assembloids, modeling the projections of the visual system. In addition, RGC survival was enhanced in long-term assembloids, overcoming prior limitations of retinal organoids in which RGCs are lost. Overall, these approaches will facilitate studies of human visual system development, as well as diseases or injuries to this critical pathway.Item Retinal Ganglion Cell Diversity and Subtype Specification from Human Pluripotent Stem Cells(Cell Press, 2018-04-10) Langer, Kirstin B.; Ohlemacher, Sarah K.; Phillips, M. Joseph; Fligor, Clarisse M.; Jiang, Peng; Gamm, David M.; Meyer, Jason S.; Biology, School of ScienceRetinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs, this class of cell is remarkably diverse, comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models, but less attention has been paid to human RGCs. Thus, efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs) and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics, confirming the combinatorial expression of molecular markers associated with these subtypes, and also provided insight into more subtype-specific markers. Thus, the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs.Item Retinal Ganglion Cells in a Dish: Current Strategies and Recommended Best Practices for Effective In Vitro Modeling of Development and Disease(Springer, 2023) Huang, Kang-Chieh; Gomes, Cátia; Meyer, Jason S.; Biology, School of ScienceThe ability to derive retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) provides an extraordinary opportunity to study the development of RGCs as well as cellular mechanisms underlying their degeneration in optic neuropathies. In the past several years, multiple approaches have been established that allow for the generation of RGCs from hPSCs, with these methods greatly improved in more recent studies to yield mature RGCs that more faithfully recapitulate phenotypes within the eye. Nevertheless, numerous differences still remain between hPSC-RGCs and those found within the human eye, with these differences likely explained at least in part due to the environment in which hPSC-RGCs are grown. With the ultimate goal of generating hPSC-RGCs that most closely resemble those within the retina for proper studies of retinal development, disease modeling, as well as cellular replacement, we review within this manuscript the current effective approaches for the differentiation of hPSC-RGCs, as well as how they have been applied for the investigation of RGC neurodegenerative diseases such as glaucoma. Furthermore, we provide our opinions on the characteristics of RGCs necessary for their use as effective in vitro disease models and importantly, how these current systems should be improved to more accurately reflect disease states. The establishment of characteristics in differentiated hPSC-RGCs that more effectively mimic RGCs within the retina will not only enable their use as effective models of RGC development, but will also create a better disease model for the identification of mechanisms underlying the neurodegeneration of RGCs in disease states such as glaucoma, further facilitating the development of therapeutic approaches to rescue RGCs from degeneration in disease states.Item Stepwise Differentiation of Retinal Ganglion Cells from Human Pluripotent Stem Cells Enables Analysis of Glaucomatous Neurodegeneration(Wiley, 2016-06) Ohlemacher, Sarah K.; Sridhar, Akshayalakshmi; Xiao, Yucheng; Hochstetler, Alexandra E.; Sarfarazi, Mansoor; Cummins, Theodore R.; Meyer, Jason S.; Department of Biology, School of ScienceHuman pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells, possess the unique ability to readily differentiate into any cell type of the body, including cells of the retina. Although previous studies have demonstrated the ability to differentiate hPSCs to a retinal lineage, the ability to derive retinal ganglion cells (RGCs) from hPSCs has been complicated by the lack of specific markers with which to identify these cells from a pluripotent source. In the current study, the definitive identification of hPSC-derived RGCs was accomplished by their directed, stepwise differentiation through an enriched retinal progenitor intermediary, with resultant RGCs expressing a full complement of associated features and proper functional characteristics. These results served as the basis for the establishment of induced pluripotent stem cells (iPSCs) from a patient with a genetically inherited form of glaucoma, which results in damage and loss of RGCs. Patient-derived RGCs specifically exhibited a dramatic increase in apoptosis, similar to the targeted loss of RGCs in glaucoma, which was significantly rescued by the addition of candidate neuroprotective factors. Thus, the current study serves to establish a method by which to definitively acquire and identify RGCs from hPSCs and demonstrates the ability of hPSCs to serve as an effective in vitro model of disease progression. Moreover, iPSC-derived RGCs can be utilized for future drug screening approaches to identify targets for the treatment of glaucoma and other optic neuropathies.