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-derived coculture 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 coculture and triculture systems, revealing distinct influences of cell types in coculture compared to when they were grown individually. We then modeled inflammatory states by activating microglia with lipopolysaccharide and evaluated their effects on RGCs both directly and in the context of astrocyte coculture. This stepwise, reductionist approach enabled us to dissect the cellular interactions driving RGC vulnerability in inflammatory conditions relevant to optic neuropathies. Our findings provide 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.

Degenerative eye diseases are major causes of irreversible vision loss worldwide, but effective treatments remain limited, partly due to the lack of effective human models. Retinal organoids derived from stem cells can recapitulate key structural and physiological features of the human retina, offering powerful tools to study disease mechanisms and develop new therapies. Here, we review recent progress in engineering retinal organoids and eye-on-a-chip models for modeling degenerative eye diseases, with a focus on engineering innovations. We first describe conventional methods for organoid differentiation and characterization along with current outstanding challenges. To better engineer retinal organoids, new strategies that leverage microfluidics and biomaterials have emerged to regulate dynamic and physiologically relevant environments for organoid differentiation. Moreover, the integration of artificial intelligence, multimodal sensing, and data analytics improves the monitoring and prediction of retinal function and therapeutic outcomes. Finally, we discuss future directions in innovating next-generation retinal organoid and eye-on-a-chip models for disease modeling, drug discovery, and vision restoration, highlighting their potential for precision ophthalmology.

Glaucoma is a complex neurodegenerative disease with multiple subtypes, yet all are characterized by the progressive dysfunction and loss of retinal ganglion cells (RGCs), which ultimately results in vision impairment and blindness. Elevated intraocular pressure (IOP) is a major risk factor for glaucoma; however, it is neither necessary nor sufficient for glaucomatous neurodegeneration, as patients can exhibit high IOP without developing glaucoma and patients can develop glaucoma with normal IOP. Yet FDA-approved treatment options are largely limited to approaches to minimize risk and reduce IOP. Thus, there is a critical need to target other aspects of glaucoma pathophysiology. Neuroinflammation is broadly defined here as immune-relevant responses, often involving microglia and astrocytes, within the central nervous system which may include peripheral immune cell infiltration. Burgeoning evidence has implicated glia in the development and progression of glaucoma in human tissues and mouse models. Most mouse models of glaucoma to date have shown that microglia and astrocytes are reactive in early stages of glaucomatous neurodegeneration prior to overt RGC loss. However, there is growing evidence that human and mouse glia adopt distinct phenotypes in response to neurodegeneration. Thus, there is critical need to expand our studies to include the new generations of human cell culture models. In this review, we discuss: 1) the evidence of neuroinflammatory processes in human glaucoma; 2) models of glaucoma relevant neuroinflammation; and the evidence specifically for 3) innate immune cell-driven and 4) macroglia-driven processes.

The National Centralized Repository for Alzheimer's Disease and Related Dementias (NCRAD) supports the etiology, early detection, and therapeutic development for Alzheimer's disease and related dementias (ADRD). One of the goals of NCRAD is to continue to offer high-quality biobanking, biospecimens, standardized ADRD biomarkers, and support for investigators utilizing cutting-edge methods and assays to advance ADRD research. NCRAD currently funds sample processing and banking for ADRD studies, and for AD Research Centers (ADRCs), supports generation of APOE genotype, plasma-based ADRD biomarkers, and supplemental GWAS data. Rigorous quality control ensures the highest quality biospecimens are processed, banked, and distributed, including: sample tracking; DNA and RNA quality measurements; hemoglobin contamination assessment; DNA fingerprinting to assess sample quality and identity; and whole genome sequencing (WGS) data generated for all banked induced pluripotent stem cell (iPSC) lines. Genetic and biomarker data is shared with data repositories including the National Institute on Aging Genetics of Alzheimer's Disease Data Storage Site (NIAGADS) and the National Alzheimer's Coordinating Center (NACC). NCRAD currently banks samples for more than 80 studies, including samples from >130,000 participants with sample types including DNA, RNA, whole blood, plasma, serum, CSF, brain tissue, stool, peripheral blood mononuclear cells (PBMCs), lymphoblast cell lines, fibroblasts, and iPSCs. More than 15,000 samples are from paired visits with more than one sample type available, and many studies bank samples from longitudinal participant visits. The repository distributed 20,000 uniform sample collection kits and received, processed, and stored more than 200,000 new aliquots in 2024. Approximately 440,000 sample aliquots have been distributed to nearly 300 researchers thus far. To date, over 1,000 publications have been generated using NCRAD samples and data. NCRAD has played a key role in development of best practices, protocol development, and uniform sample collection for ADRD studies. NCRAD continues to support cutting-edge research in genetics, genomics, and biomarker research and, more recently, implementation of the Alzheimer's Association Revised Criteria for diagnosing AD by making plasma biomarker data broadly available for ADRC participants. As such, NCRAD has and continues to play a vital part in the advancement of translational ADRD research.

Reduction–oxidation factor-1 or apurinic/apyrimidinic endonuclease 1 (Ref-1/APE1) is a crucial redox-sensitive activator of transcription factors such as NF-κB, HIF-1α, STAT-3 and others. It could contribute to key features of ocular neovascularization including inflammation and angiogenesis; these underlie diseases like neovascular age-related macular degeneration (nAMD). We previously revealed a role for Ref-1 in the growth of ocular endothelial cells and in choroidal neovascularization (CNV). Here, we set out to further explore Ref-1 in neovascular eye disease. Ref-1 was highly expressed in human nAMD, murine laser-induced CNV and Vldlr−/− mouse subretinal neovascularization (SRN). Ref-1’s interaction with a redox-specific small molecule inhibitor, APX2009, was shown by NMR and docking. This compound blocks crucial angiogenic features in multiple endothelial cell types. APX2009 also ameliorated murine laser-induced choroidal neovascularization (L-CNV) when delivered intravitreally. Moreover, systemic APX2009 reduced murine SRN and downregulated the expression of Ref-1 redox regulated HIF-1α target carbonic anhydrase 9 (CA9) in the Vldlr−/− mouse model. Our data validate the redox function of Ref-1 as a critical regulator of ocular angiogenesis, indicating that inhibition of Ref-1 holds therapeutic potential for treating nAMD.

INTRODUCTION:Variants of phospholipase C gamma 2 (PLCG2), a key microglial immune signaling protein, are genetically linked to Alzheimer's disease (AD) risk. Understanding how PLCG2 variants alter microglial function is critical for identifying mechanisms that drive neurodegeneration or resiliency in AD. METHODS:Induced pluripotent stem cell (iPSC) -derived microglia carrying the protective PLCG2P522R or risk-conferring PLCG2M28L variants, or loss of PLCG2, were generated to ascertain the impact on microglial transcriptome and function. RESULTS:Protective PLCG2P522R microglia showed significant transcriptomic similarity to isogenic controls. In contrast, risk-conferring PLCG2M28L microglia shared similarities with PLCG2KO microglia, with functionally reduced TREM2 expression, blunted inflammatory responses, and increased proliferation and cell death. Uniquely, PLCG2P522R microglia showed elevated cytokine secretion after lipopolysaccharide (LPS) stimulation and were protected from apoptosis. DISCUSSION:These findings demonstrate that PLCG2 variants drive distinct microglia transcriptomes that influence microglial functional responses that could contribute to AD risk and protection. Targeting PLCG2-mediated signaling may represent a powerful therapeutic strategy to modulate neuroinflammation. HIGHLIGHTS:The impact of Alzheimer's disease protective- and risk-associated variants of phospholipase C gamma 2 (PLCG2) on the transcriptome and function of induced pluripotent stem cell (iPSC) -derived microglia was investigated. PLCG2 risk variant microglia exhibited a basal transcriptional profile similar to PLCG2-deficient microglia but significantly different from isotype control and the transcriptionally similar PLCG2 protective variant microglia. PLCG2 risk variant and PLCG2-deficient microglia show decreased levels of triggering receptor expressed on myeloid cells 2 (TREM2). The differential transcriptional pathways of protective and risk-associated PLCG2 variant microglia functionally affect proliferation, apoptosis, and immune response. Protective PLCG2 microglia show resilience to apoptosis and increased cytokine/chemokine secretion upon exposure to lipopolysaccharide (LPS).

Human vision relies on retinal ganglion cells (RGCs), and their connectivity with distinct brain regions enables higher order visual processing. RGCs vary considerably between species, and small model organisms display distinct RGC innervation patterns from that in humans. There is thus a need for robust models of human RGC circuit formation that preserves innervation specificity. Here, we developed an in vitro microfluidics eye-to-brain connectivity model using human pluripotent stem cell (hPSC)-derived RGCs to assess brain region-specific connectivity features. We find that cultured human RGCs segregate their dendrites and axons and display axonal features that align with that of their in vivo human RGC counterparts. The addition of region-specific brain targets to human RGC axons terminals resulted in differential RGC connectivity with distinct retinorecipient brain regions. Increased synapse formation occurred between RGCs and lateral geniculate neurons relative to that with superchiasmatic nucleus neurons, mirroring in vivo innervation differences. Both retinorecipient partners induced the formation of more synapses relative to non-retinorecipient brain target controls. These results suggest that human RGC innervation properties are preserved in culture systems and that human RGCs can differentially sense and respond to retinorecipient targets to control wiring outcomes. These systems may aid in the discovery of human-specific wiring factors for potential therapeutic applications. Significance Statement:This study presents a novel in vitro model to investigate human retinal ganglion cell (RGC) connectivity, using human pluripotent stem cell-derived RGCs. By modeling human eye-to-brain connections in a microfluidic devise, this system reveals how human RGCs form selective, region-specific synapses with brain areas like the lateral geniculate nucleus and the suprachiasmatic nucleus. The findings demonstrate that human RGCs retain their innervation specificity presences in culture, mimicking in vivo human connectivity patterns. This model provides a powerful tool for understanding the factors controlling human-specific brain wiring, with potential applications in therapies for visual and neurological disorders.

The National Centralized Repository for Alzheimer’s Disease and Related Dementias (NCRAD) is continuing to develop a bank of induced pluripotent stem cells (iPSCs) that are available by request to the Alzheimer’s disease (AD) research community. As part of the pipeline for quality control of received cell lines, DNA was extracted for all lines and was submitted for whole genome sequencing (WGS). Paired-end WGS data was generated using the Illumina NovaSeq 6000 and processed following GATK best practices using the Sentieon pipeline. WGS data was annotated with Annovar, and data was reviewed for reported cell line variants and checked with Varsome and Franklin. Sequencing data was reviewed for all nonsynonymous and splicing variants in the APP , PSEN1 , PSEN2 , GRN , and MAPT genes. Additionally, DNA from cell lines was genotyped in-house by NCRAD to generate apolipoprotein E (APOE) genotypes, and this data was compared with the WGS to confirm sample identity. Basic clinical and demographic data was also collected, including sex, case/control status, age, race, and ethnicity. To date, DNA has been extracted and genotyped at NCRAD for lines from 183 participants including generation of APOE genotypes passing quality control. Of these, 120 cell lines have returned WGS data passing quality control. Table 1 describes the demographic and clinical features for these lines, which include data for lines from 90 individuals as well as data for 30 isogenic lines. Of the 120 lines with available WGS, there are 13 case APP variant carriers, 13 case MAPT variant carriers, 8 case PSEN1 variant carriers, and 2 case PSEN2 variant carriers. Additionally, these cell lines included two control carriers of variants of uncertain significance (VUS) in GRN or PSEN2 , as well as two cases carrying VUS in PSEN1 or APP . NCRAD continues to expand iPSCs for the research community; adding WGS data to this resource provides an expanded scope for pre-screening as well as functional research. Future directions include review of variants being tested in the Model Organism Development & Evaluation for Late-Onset AD (MODEL-AD) to provide additional value to researchers.

Dysfunctional microglial activity has recently been identified as a potential mechanism leading to accumulation of amyloid beta and pTau and subsequent neurodegeneration in Alzheimer’s Disease. The CX3CR1/fractalkine axis serves as a mechanism for bi-directional communication between microglia and neurons, respectively, to promote a resting, anti-inflammatory state in microglia. Previous studies have demonstrated that deficiency in CX3CR1 signaling leads microglia to a more pro-inflammatory phenotype, phagocytic deficits, and increased susceptibility of neurons to cell death. Additionally, the CX3CR1-V249I polymorphism was recently identified as a potential risk allele for Alzheimer’s Disease with worsened Braak staging in post-mortem Alzheimer’s patients. However, the role of fractalkine dysfunction in human cells and the mechanisms by which microglia with the CX3CR1-V249I SNP contribute to neurodegeneration remain unclear. To address this shortcoming, we utilized human induced pluripotent stem cells and CRISPR/Cas9 gene editing technology to elucidate the effects of the CX3CR1-V249I polymorphism on human microglia-like cells (hMGLs) compared to an isogenic control cell line. Isogenic control cells alongside both heterozygous and homozygous CX3CR1 V249I cell lines were differentiated in parallel to yield enriched populations of hMGLs. Resulting hMGLs were then assessed for uptake of amyloid beta 1-42 using flow cytometry, cell death in response to cytokine starvation, changes in proliferation, and finally alterations to migratory behavior using a microfluidic chamber. We demonstrate the effective differentiation of hMGLS from both isogenic control and CX3CR1-V249I backgrounds, which express characteristic microglial markers and are functionally phagocytic. Microglia bearing the homozygous CX3CR1-V249I allele, but not heterozygous cells, demonstrated decreased uptake of amyloid beta in vitro compared to isogenic controls. Additionally, homozygous V249I microglia demonstrated increased stress-induced cell death, as well as altered proliferation and decreased migratory capability. These findings suggest that the CX3CR1-V249I polymorphism may cause a dysfunctional microglia phenotype that may contribute to neuronal dysfunction and death. Ongoing work will expand upon the transcriptome and secretome profile of CX3CR1-V249I microglia and elucidate how this gene variant contributes to Alzheimer’s Disease-related neurodegeneration.

Retinal ganglion cells (RGCs) are highly compartmentalized neurons whose long axons serve as the sole connection between the eye and the brain. In both injury and disease, RGC degeneration occurs in a similarly compartmentalized manner, with distinct molecular and cellular responses in the axonal and somatodendritic regions. The goal of this study was to establish a microfluidic-based platform to investigate RGC compartmentalization in both health and disease states. Human pluripotent stem cell (hPSC)-derived RGCs were seeded into microfluidic devices that allow physical separation of axons from the somatodendritic compartment, enabling precise study of each region. Initial experiments characterized axonal outgrowth and the specific segregation of axons and dendrites. We then examined compartment-specific phenotypes in RGCs carrying the OPTN(E50K) glaucoma mutation compared to isogenic controls, including differences in axonal growth and axonal transport efficiency, with OPTN-mutant RGCs showing reduced axon length and slower transport, hallmarks of neurodegeneration. Axonal RNA-seq analyses revealed transcriptomic alterations related to disease states, including specific transcriptomic changes along OPTN axons. To assess glial influences on axonal health, we developed models with astrocytes localized specifically to the proximal axonal compartment and modulated their disease states to simulate pathological conditions. Importantly, the induction of diseased astrocytes solely along proximal axons triggered compartment-specific neurodegenerative changes in RGCs. Collectively, this platform represents a successful recapitulation of the spatially distinct features of hPSC-derived RGCs under both healthy and disease conditions, offering a physiologically relevant, human-specific in vitro system to study neuronal development, axon-glia interactions, and mechanisms underlying neurodegeneration.

The involvement of microglia in neurodegenerative diseases has drawn increasing attention, as many genetic risk factors are preferentially expressed in microglia. Microglial fractalkine receptor (CX3CR1) signaling regulates many key microglial functions, and the CX3CR1-V249I single nucleotide polymorphism (SNP) has been associated with increased risk for multiple neurodegenerative conditions, including Alzheimer's disease, yet its functional consequences in human microglia remain unexplored. In this study, we generated iPSC-derived human microglia-like cells (hMGLs) and found that the CX3CR1-V249I variant increased susceptibility to starvation-induced cell death, reduced amyloid-beta uptake, altered microglial morphology, and impaired migration, with more pronounced effects in homozygous cells. Co-culture with neurons demonstrated that hMGLs with the CX3CR1-V249I variant misregulated neuronal properties, including abnormal neuronal growth as well as an induction of neuronal hyperexcitability. These findings highlight the critical role of CX3CR1 in regulating microglial function and implicate the V249I variant in driving pathogenic microglial states relevant to neurodegeneration.

The ability to derive retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) has led to numerous advances in the field of retinal research, with great potential for the use of hPSC-derived RGCs for studies of human retinal development, in vitro disease modeling, drug discovery, as well as their potential use for cell replacement therapeutics. Of all these possibilities, the use of hPSC-derived RGCs as a human-relevant platform for in vitro disease modeling has received the greatest attention, due to the translational relevance as well as the immediacy with which results may be obtained compared to more complex applications like cell replacement. While several studies to date have focused upon the use of hPSC-derived RGCs with genetic variants associated with glaucoma or other optic neuropathies, many of these have largely described cellular phenotypes with only limited advancement into exploring dysfunctional cellular pathways as a consequence of the disease-associated gene variants. Thus, to further advance this field of research, in the current study we leveraged an isogenic hPSC model with a glaucoma-associated mutation in the Optineurin (OPTN) protein, which plays a prominent role in autophagy. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor AMPK, along with subsequent neurodegeneration in OPTN(E50K) RGCs differentiated from hPSCs, and have further validated some of these findings in a mouse model of ocular hypertension. 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 highlighted that autophagy disruption resulted in increased autophagic demand which was associated with downregulated signaling through mTORC1, contributing to the degeneration of RGCs.

Reactive 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.

Human 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.

The iDA Project (iPSCs to Study Diversity in Alzheimer’s and Alzheimer’s Disease-related Dementias) is generating 200 induced pluripotent stem cell lines from Alzheimer’s Disease Neuroimaging Initiative participants. These lines are sex balanced, include common APOE genotypes, span disease stages, and are ancestrally diverse. Cell lines and characterization data will be shared openly.