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A human pluripotent stem cell tri-culture platform to elucidate microglial regulation of retinal ganglion cells in neuroinflammation

ABSTRACT: Optic neuropathies, including glaucoma, are characterized by the progressive degeneration of retinal ganglion cells (RGCs), ultimately leading to irreversible vision loss. Increasing evidence implicates microglia, the resident immune cells of the central nervous system, as key modulators of RGC health and disease progression. However, the precise mechanisms by which microglia influence RGCs remain poorly understood, particularly in the human context. In this study, we established human pluripotent stem cell (hPSC)-derived co-culture systems incorporating microglia, astrocytes, and RGCs to explore how microglia shape RGC growth and maturation under physiological conditions. We first examined the impact of homeostatic microglia on RGCs in both co-culture and tri-culture systems, revealing distinct influences of cell types in co-culture compared to when they were grown individually. We then modeled inflammatory states by activating microglia with lipopolysaccharide (LPS) and evaluated their effects on RGCs both directly and in the context of astrocyte co-culture. This stepwise, reductionist approach enabled us to dissect the cellular interactions driving RGC vulnerability in inflammatory conditions relevant to optic neuropathies. Our findings provide new insight into the complex neuroimmune landscape that underlies RGC degeneration and identify key pathways that may serve as therapeutic targets across a range of optic nerve diseases

ORGANISM(S): Homo sapiens

PROVIDER: GSE308054 | GEO | 2025/09/20

REPOSITORIES: GEO

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The efficient induction of human retinal ganglion-like cells provides a platform for studying optic neuropathies

Project description:Retinal ganglion cells (RGCs) are essential for vision perception. In glaucoma and other optic neuropathies, RGCs and their optic axons undergo degenerative change and cell death; this can result in irreversible vision loss. Here we developed a rapid protocol for directly inducing RGC differentiation from human induced pluripotent stem cells (iPSCs) by the overexpression of ATOH7, BRN3B and SOX4. The hiPSC-derived RGC-like cells (iRGCs) show robust expression of various RGC-specific markers, such as BRN3A, EBF1, ISL1 and RBPMS. A functional assessment was also carried out and this demonstrated that these iRGCs display stimulus-induced neuronal activity, as well as spontaneous neuronal activity. Ethambutol (EMB), an effective first-line anti-tuberculosis agent, is known to cause serious visual impairment and irreversible vision loss due to the RGC degeneration in a significant number of treated patients. Using our iRGCs, EMB was found to induce significant dose-dependent and time-dependent increases in cell death and neurite degeneration. Western blot analysis revealed that the expression levels of p62 and LC3-II were upregulated, and further investigations revealed that EMB caused a blockade of lysosome-autophagosome fusion; this indicates that impairment of autophagic flux is one of the adverse effects of that EMB has on iRGCs. In addition, EMB was found to elevate intracellular reactive oxygen species (ROS) levels increasing apoptotic cell death. This could be partially rescued by the co-treatment with the ROS scavenger NAC. Taken together, our findings suggest that this iRGC model, which achieves both high yield and high purity, is suitable for investigating optic neuropathies, as well as being useful when searching for potential drugs for therapeutic treatment and/or disease prevention.

2023-08-04 | GSE236027 | GEO

Global gene expression changes in rat retinal ganglion cells after experimental glaucoma

Project description:Goals of the study: 1. Assess the gene expression profile in rat RGC after experimental IOP elevation to elucidate the molecular mechanisms of RGC death. 2. Identify potentially novel genes and pathways that may contribute to RGC death in glaucoma. Background: Glaucoma is a neurodegenerative disease characterized by the slow, progressive degeneration of RGC. Elevated IOP is the biggest risk factor for developing glaucoma, loss of RGC and optic nerve atrophy. The pathophysiology of glaucomatous neurodegeneration is not fully understood. It is now clear that RGC die by apoptosis in glaucoma. However, what triggers the apoptosis is still unknown. So far, several gene expression studies have been performed on the retina as a whole. These studies revealed up and downregulation of many genes in response to elevated IOP and optic nerve transection. However, the retina is a complex tissue composed of neuronal, glial and vascular cell types. The RGCs only comprise 5% or less of retinal cells. The gene expression profiles from whole retina can not represent of RGC gene expression. In the current study, we sought to investigate the whole genome regulation of the RGCs in glaucoma. The study was carried out in 3 adult male Brown Norway rats with experimental glaucoma. An equal number of RGCs were captured from normal eyes and eyes with elevated IOP. Gene expression in the glaucomatous RGC was compared with that in the fellow RGC by using Affymetrix Gene chip.

2010-10-01 | E-GEOD-12596 | biostudies-arrayexpress

MicroRNA-19a-PTEN axis is involved in the developmental decline of axon regenerative capacity in retinal ganglion cells

Project description:Irreversible blindness from glaucoma and optic neuropathies is attributed to retinal ganglion cells (RGCs) losing the ability to regenerate axons. While several transcription factors and proteins have demonstrated enhancement of axon regeneration after optic nerve injury, mechanisms contributing to the age-related decline in axon regenerative capacity remains elusive. Here, we show that microRNAs are differentially expressed during RGC development, and identify microRNA-19a (miR-19a) as a heterochronic marker; developmental decline of miR-19a relieves suppression of PTEN, a key regulator of axon regeneration, and serves as a temporal indicator of decreasing axon regenerative capacity. Intravitreal injection of miR-19a promotes axon regeneration after optic nerve crush in adult mice, and increases axon extension in RGCs isolated from aged human donors. This uncovers a previously unrecognized involvement of the miR-19a-PTEN axis in RGC axon regeneration, and demonstrates therapeutic potential of microRNA-mediated restoration of axon regenerative capacity via intravitreal injection in patients with optic neuropathies.

2020-07-04 | GSE102458 | GEO

Waves of cell death modulate the abundance and maturation of human retinal ganglion cells

Project description:Retinal ganglion cells (RGCs) are the projection neurons that transmit visual information from the retina to the brain via the optic nerve. A substantial proportion of RGCs are eliminated by programmed cell death during development to regulate their final number, yet how cell death impacts RGC development in human retinas remains poorly understood. Here, we characterized the timing and cell-type-specificity of cell death in human fetal retinas and retinal organoids. We identified two waves of apoptosis: an early wave targeting retinal neuronal precursors and a late wave affecting RGCs and other neurons. In addition to these two waves of apoptosis, organoids displayed a distinct wave of necrosis in the innermost core region that eliminates nearly all RGCs during long-term culture. To investigate the functions of the apoptotic waves during retinal development, we differentiated BAX/BAK double mutant organoids that are deficient for apoptosis. In these mutant organoids, RGC survival was improved, RGC neurogenesis and maturation were delayed, and RGCs were lost through a compensatory increase in necrosis. Our data suggest that the waves of developmental apoptosis promote human RGC neurogenesis and maturation. Together, our results highlight the roles of cell death in human RGC development and the challenges in retinal organoid design. Overcoming these obstacles would improve the utility of retinal organoids for modeling optic neuropathies like glaucoma

2025-11-02 | GSE305194 | GEO

Genome wide chromatin accessibility analysis reveals a role for CREB in retinal ganglion cells axon growth decline in development and regeneration after optic nerve injury [RNA-seq]

Project description:CNS neurons lose their ability to grow and regenerate axons during development. This is the case for Retinal Ganglion Cells (RGCs) in the retina, which transmit visual information to the brain via axons projecting into the optic nerve. RGCs are unable to regenerate their axon after injury, and start a degeneration process that leads to cell death and loss of vision. To identifying molecular mechanisms that increase regeneration of RGC and may offer new treatment strategies for patients with glaucoma or other types of optic neuropathies, we focused on the identification of transcription factors and chromatin accessible sites that are enriched in RGC during developmental stages, in which axon growth capacity is robust. We find that stage-specific gene expression changes are correlated with temporal changes in promoter chromatin accessibility. We also find that Creb binding motifs are enriched in the differentially opened regions of the chromatin at embryonic developmental stage. Overexpression of active Creb promotes axon regeneration after optic nerve injury. Our results provide a map of the chromatin accessibility during RGC development and highlights that manipulating TF associated with developmental stages can stimulate axon growth in adulthood.

2021-07-28 | GSE163563 | GEO

Genome wide chromatin accessibility analysis reveals a role for CREB in retinal ganglion cells axon growth decline in development and regeneration after optic nerve injury [ATAC-seq]

2021-07-28 | GSE163562 | GEO

Transcriptomic profiling of retinal cells reveals a subpopulation of microglia/macrophages expressing Rbpms marker of retinal ganglion cells (RGCs) that confound identification of RGCs

Project description:Analysis of retinal ganglion cells (RGCs) by scRNA-seq is emerging as a state-of-the-art method for studying RGC biology and subtypes, as well as for studying the mechanisms of neuroprotection and axon regeneration in the central nervous system (CNS). Rbpms has been established as a pan-RGC marker, and Spp1 has been established as an αRGC type and macrophage marker. Here, we analyzed by scRNA-seq retinal microglia and macrophages, and found Rbpms+ subpopulations of retinal microglia/macrophages, which pose a potential pitfall in scRNA-seq studies involving RGCs. We performed comparative analysis of cellular identity of the presumed RGC cells isolated in recent scRNA-seq studies, and found that Rbpms+ microglia/macrophages confounded identification of RGCs. We also showed using immunohistological analysis that, Rbpms protein localizes to stress granules in a subpopulation of retinal microglia after optic nerve injury, which was further supported by bioinformatics analysis identifying stress granule-associated genes enriched in the Rbpms+ microglia/macrophages. Our findings suggest that the identification of Rbpms+ RGCs by immunostaining after optic nerve injury should exclude cells in which Rbpms signal is restricted to a subcellular granule, and include only those cells in which the Rbpms signal is labeling cell soma diffusely. Finally, we provide solutions for circumventing this potential pitfall of Rbpm-expressing microglia/macrophages in scRNA-seq studies, by including in RGC and αRGC selection criteria other pan-RGC and αRGC markers.

2023-05-23 | GSE228986 | GEO

Direct-induced human retinal ganglion cells mimic fetal RGCs and are neuroprotective after transplant in vivo

Project description:Since retinal ganglion cells (RGCs) do not regenerate after injury, RGC replacement therapy could provide an approach to vision restoration in glaucoma and other optic neuropathies. Here we developed a rapid protocol for direct induction of RGC (iRGC) differentiation from human iPSCs and ESCs in less than two weeks, by overexpression of NGN2 in RGC survival-promoting medium supplemented with a Notch inhibitor. Neuronal morphology and neurite growth were observed within one week of induction. Immunostaining and qRT-PCR for characteristic RGC-specific genes confirmed identity. Calcium imaging was used to evaluate iRGCs’ electrophysiologic maturation, and demonstrated GABA-induced excitatory calcium influx characteristic of immature RGCs. Using single cell-RNA sequencing (scRNA-seq) we further delineated iRGCs’ differentiation and compared iRGC transcriptomic profiles to fetal human and retinal organoid-derived RGCs. Unbiased clustering showed high similarities of transcriptomic profiles among iRGCs, early-stage fetal human RGCs and early-stage retinal organoid-derived RGCs. However, for some markers, including BRN3a, BRN3b and NEFL, iRGCs demonstrated expression patterns more similar to fetal RGCs than to retinal organoid RGCs. After intravitreal injection into rodent eyes, transplanted iRGCs survived and migrated into host retinas where they were detected one week and one month after transplant. Prior optic nerve trauma to the recipient host did not significantly enhance or detract from iRGC integration, but iRGCs protected host RGCs from neurodegeneration. Taken together, these data demonstrate rapid iRGC generation in vitro into an immature cell with high similarity to human fetal RGCs and capacity for retinal integration after transplant, including after optic nerve injury. The simplicity of this system may benefit translational studies on human RGCs.

2022-04-10 | GSE200452 | GEO

Displacement of PDE4D3 with an anchoring disruptor peptide is RGC protective after injury

Project description:Loss of retinal ganglion cells (RGCs) is central to the pathogenesis of optic neuropathies such as glaucoma. Increased cAMP signaling in RGCs is neuroprotective, as has been previously demonstrated in multiple animal models, including optic nerve crush (ONC) injury. We have shown that displacement of the cAMP-specific phosphodiesterase PDE4D3 from an RGC perinuclear compartment by expression of the modified PDE4D3 N-terminal peptide 4D3(E) increases perinuclear protein kinase A activity in cultured neurons and RGC survival in vivo after ONC injury. To explore potential mechanisms by which 4D3(E) expression promotes neuroprotection, mice intravitreally injected with an adeno-associated virus to express an mCherry-tagged 4D3(E) peptide were subjected to ONC injury and analyzed by single cell RNA-sequencing (scRNA-seq) for changes in RGC gene expression. 4D3(E)-mCherry expression was associated with an attenuation of injury-induced changes in gene expression, thereby supporting the hypothesis that enhanced perinuclear PKA signaling promotes neuroprotective RGC gene expression.

2024-08-28 | GSE260952 | GEO

Differential gene expression and pathway analysis by RNA sequencing in human OPTN(E50K) astrocytes

Project description:Retinal ganglion cell (RGC) degeneration is a primary characteristic of glaucoma, although non-cell autonomous mechanisms have been implicated in RGC dysfunction. Astrocytes closely associate with RGCs in the nerve fiber layer of the retina and optic nerve, where they can contribute to RGC neurodegeneration. However, the mechanisms by which astrocytes promote neurotoxicity and contribute to glaucoma remain unclear. Here we present data describing disease phenotypes in astrocytes and their ability to modulate RGC health.

2021-04-23 | GSE173129 | GEO

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