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Browsing by Author "Huang, Kang-Chieh"
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Item A highly reproducible and efficient method for retinal organoid differentiation from human pluripotent stem cells(National Academy of Sciences, 2024) Harkin, Jade; Peña, Kiersten H.; Gomes, Cátia; Hernandez, Melody; Lavekar, Sailee S.; So, Kaman; Lentsch, Kelly; Feder, Elyse M.; Morrow, Sarah; Huang, Kang-Chieh; Tutrow, Kaylee D.; Morris, Ann; Zhang, Chi; Meyer, Jason S.; Pharmacology and Toxicology, School of MedicineHuman pluripotent stem cell (hPSC)-derived retinal organoids are three-dimensional cellular aggregates that differentiate and self-organize to closely mimic the spatial and temporal patterning of the developing human retina. Retinal organoid models serve as reliable tools for studying human retinogenesis, yet limitations in the efficiency and reproducibility of current retinal organoid differentiation protocols have reduced the use of these models for more high-throughput applications such as disease modeling and drug screening. To address these shortcomings, the current study aimed to standardize prior differentiation protocols to yield a highly reproducible and efficient method for generating retinal organoids. Results demonstrated that through regulation of organoid size and shape using quick reaggregation methods, retinal organoids were highly reproducible compared to more traditional methods. Additionally, the timed activation of BMP signaling within developing cells generated pure populations of retinal organoids at 100% efficiency from multiple widely used cell lines, with the default forebrain fate resulting from the inhibition of BMP signaling. Furthermore, given the ability to direct retinal or forebrain fates at complete purity, mRNA-seq analyses were then utilized to identify some of the earliest transcriptional changes that occur during the specification of these two lineages from a common progenitor. These improved methods also yielded retinal organoids with expedited differentiation timelines when compared to traditional methods. Taken together, the results of this study demonstrate the development of a highly reproducible and minimally variable method for generating retinal organoids suitable for analyzing the earliest stages of human retinal cell fate specification.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 Autophagy disruption reduces mTORC1 activation leading to retinal ganglion cell neurodegeneration associated with glaucoma(Cold Spring Harbor Laboratory, 2023-01-04) Huang, Kang-Chieh; Gomes, Cátia; Shiga, Yukihiro; Belforte, Nicolas; VanderWall, Kirstin B.; Lavekar, Sailee S.; Fligor, Clarisse M.; Harkin, Jade; Di Polo, Adriana; Meyer, Jason S.; Biology, School of ScienceAutophagy dysfunction has been associated with several neurodegenerative diseases including glaucoma, characterized by the degeneration of retinal ganglion cells (RGCs). However, the mechanisms by which autophagy dysfunction promotes RGC damage remain unclear. Here, we hypothesized that perturbation of the autophagy pathway results in increased autophagic demand, thereby downregulating signaling through mammalian target of rapamycin complex 1 (mTORC1), a negative regulator of autophagy, contributing to the degeneration of RGCs. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor adenosine monophosphate-activated protein kinase (AMPK), along with subsequent neurodegeneration in RGCs differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated variant of Optineurin (OPTN-E50K). Similarly, the microbead occlusion model of glaucoma resulting in ocular hypertension also exhibited autophagy disruption and mTORC1 downregulation. Pharmacological inhibition of mTORC1 in hPSC-derived RGCs recapitulated disease-related neurodegenerative phenotypes in otherwise healthy RGCs, while the mTOR-independent induction of autophagy reduced protein accumulation and restored neurite outgrowth in diseased OPTN-E50K RGCs. Taken together, these results highlight an important balance between autophagy and mTORC1 signaling essential for RGC homeostasis, while disruption to these pathways contributes to neurodegenerative features in glaucoma, providing a potential therapeutic target to prevent neurodegeneration.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 Enhanced mitochondrial biogenesis promotes neuroprotection in human pluripotent stem cell derived retinal ganglion cells(Springer Nature, 2023-02-24) Surma, Michelle; Anbarasu, Kavitha; Dutta, Sayanta; Olivera Perez, Leonardo J.; Huang, Kang-Chieh; Meyer, Jason S.; Das, Arupratan; Ophthalmology, School of MedicineMitochondrial dysfunctions are widely afflicted in central nervous system (CNS) disorders with minimal understanding on how to improve mitochondrial homeostasis to promote neuroprotection. Here we have used human stem cell differentiated retinal ganglion cells (hRGCs) of the CNS, which are highly sensitive towards mitochondrial dysfunctions due to their unique structure and function, to identify mechanisms for improving mitochondrial quality control (MQC). We show that hRGCs are efficient in maintaining mitochondrial homeostasis through rapid degradation and biogenesis of mitochondria under acute damage. Using a glaucomatous Optineurin mutant (E50K) stem cell line, we show that at basal level mutant hRGCs possess less mitochondrial mass and suffer mitochondrial swelling due to excess ATP production load. Activation of mitochondrial biogenesis through pharmacological inhibition of the Tank binding kinase 1 (TBK1) restores energy homeostasis, mitigates mitochondrial swelling with neuroprotection against acute mitochondrial damage for glaucomatous E50K hRGCs, revealing a novel neuroprotection mechanism.Item Exploring dysfunctional barrier phenotypes associated with glaucoma using a human pluripotent stem cell-based model of the neurovascular unit(Springer Nature, 2024-11-14) Lavekar, Sailee S.; Hughes, Jason M.; Gomes, Cátia; Huang, Kang-Chieh; Harkin, Jade; Canfield, Scott G.; Meyer, Jason S.; Biology, School of ScienceGlaucoma is a neurodegenerative disease that results in the degeneration of retinal ganglion cells (RGCs) and subsequent loss of vision. While RGCs are the primary cell type affected in glaucoma, neighboring cell types selectively modulate RGCs to maintain overall homeostasis. Among these neighboring cell types, astrocytes, microvascular endothelial cells (MVECs), and pericytes coordinate with neurons to form the neurovascular unit that provides a physical barrier to limit the passage of toxic materials from the blood into neural tissue. Previous studies have demonstrated that these barrier properties may be compromised in the progression of glaucoma, yet mechanisms by which this happens have remained incompletely understood. Thus, the goals of this study were to adapt a human pluripotent stem cell (hPSC)-based model of the neurovascular unit to the study of barrier integrity relevant to glaucoma. To achieve this, hPSCs were differentiated into the cell types that contribute to this barrier, including RGCs, astrocytes, and MVECs, then assembled into an established Transwell®-insert model. The ability of these cell types to contribute to an in vitro barrier model was tested for their ability to recapitulate characteristic barrier properties. Results revealed that barrier properties of MVECs were enhanced when cultured in the presence of RGCs and astrocytes compared to MVECs cultured alone. Conversely, the versatility of this system to model aspects of barrier dysfunction relevant to glaucoma was tested using an hPSC line with a glaucoma-specific Optineurin (E50K) mutation as well as a paired isogenic control, where MVECs then exhibited reduced barrier integrity. To identify factors that could result in barrier dysfunction, results revealed an increased expression of TGFβ2 in glaucoma-associated OPTN(E50K) astrocytes, indicating a potential role for TGFβ2 in disease manifestation. To test this hypothesis, we explored the ability to modulate exogenous TGFβ2 in both isogenic control and OPTN(E50K) experimental conditions. Collectively, the results of this study indicated that the repurposing of this in vitro barrier model for glaucoma reliably mimicked some aspects of barrier dysfunction, and may serve as a platform for drug discovery, as well as a powerful in vitro model to test the consequences of barrier dysfunction upon RGCs in glaucoma.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 Induction of astrocyte reactivity promotes neurodegeneration in human pluripotent stem cell models(Elsevier, 2024) Gomes, Cátia; Huang, Kang-Chieh; Harkin, Jade; Baker, Aaron; Hughes, Jason M.; Pan, Yanling; Tutrow, Kaylee; VanderWall, Kirstin B.; Lavekar, Sailee S.; Hernandez, Melody; Cummins, Theodore R.; Canfield, Scott G.; Meyer, Jason S.; Medical and Molecular Genetics, School of MedicineReactive astrocytes are known to exert detrimental effects upon neurons in several neurodegenerative diseases, yet our understanding of how astrocytes promote neurotoxicity remains incomplete, especially in human systems. In this study, we leveraged human pluripotent stem cell (hPSC) models to examine how reactivity alters astrocyte function and mediates neurodegeneration. hPSC-derived astrocytes were induced to a reactive phenotype, at which point they exhibited a hypertrophic profile and increased complement C3 expression. Functionally, reactive astrocytes displayed decreased intracellular calcium, elevated phagocytic capacity, and decreased contribution to the blood-brain barrier. Subsequently, co-culture of reactive astrocytes with a variety of neuronal cell types promoted morphological and functional alterations. Furthermore, when reactivity was induced in astrocytes from patient-specific hPSCs (glaucoma, Alzheimer's disease, and amyotrophic lateral sclerosis), the reactive state exacerbated astrocytic disease-associated phenotypes. These results demonstrate how reactive astrocytes modulate neurodegeneration, significantly contributing to our understanding of a role for reactive astrocytes in neurodegenerative diseases.Item Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis(Elsevier, 2019-11-12) Huang, Kang-Chieh; Wang, Mong-Lien; Chen, Shih-Jen; Kuo, Jean-Cheng; Wang, Won-Jing; Nguyen, Phan Nguyen Nhi; Wahlin, Karl J.; Lu, Jyh-Feng; Tran, Audrey A.; Shi, Michael; Chien, Yueh; Yarmishyn, Aliaksandr A.; Tsai, Ping-Hsing; Yang, Tien-Chun; Jane, Wann-Neng; Chang, Chia-Ching; Peng, Chi-Hsien; Schlaeger, Thorsten M.; Chiou, Shih-Hwa; Biology, School of ScienceX-linked juvenile retinoschisis (XLRS), linked to mutations in the RS1 gene, is a degenerative retinopathy with a retinal splitting phenotype. We generated human induced pluripotent stem cells (hiPSCs) from patients to study XLRS in a 3D retinal organoid in vitro differentiation system. This model recapitulates key features of XLRS including retinal splitting, defective retinoschisin production, outer-segment defects, abnormal paxillin turnover, and impaired ER-Golgi transportation. RS1 mutation also affects the development of photoreceptor sensory cilia and results in altered expression of other retinopathy-associated genes. CRISPR/Cas9 correction of the disease-associated C625T mutation normalizes the splitting phenotype, outer-segment defects, paxillin dynamics, ciliary marker expression, and transcriptome profiles. Likewise, mutating RS1 in control hiPSCs produces the disease-associated phenotypes. Finally, we show that the C625T mutation can be repaired precisely and efficiently using a base-editing approach. Taken together, our data establish 3D organoids as a valid disease model.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.