Enhancing human iPSC-derived retinal organoids by incorporating microglia-like cells
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ABSTRACT:
PROVIDER: PRJNA724040 | ENA |
REPOSITORIES: ENA
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Project description:Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning, which makes them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions can trigger neurodegeneration exacerbating the effect of the disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite their close resemblance to the in vivo retina, they lack some key physiological features including immune cells. To enhance the retinal organoid model, we engineered an hiPSC co-culture system containing the hiPSC-derived retinal organoids and hiPSC-derived microglia-like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 21.5 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co-cultured with the organoids. Single cell RNA-Seq transcriptomic analyses, protein expression, electron microscopy imaging and electrophysiological recordings showed that retinal organoids developed normally, expressed key markers, and retained their ability to generate spiking activity in response to light. Thus, this new co-culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina.
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Project description:Microglia are the tissue-resident macrophages of the retina and brain, being critically involved in organ development, tissue homeostasis, and response to cellular damage. Until now, little is known about the transcriptional profile of human retinal microglia and how they differentiate from peripheral monocytes, as well as from brain microglia. Additionally, the degree to which mice are suitable models for human retinal microglia is still not clear. The present study applies fluorescence-activated cell sorting to isolate human retinal microglia from enucleated eyes and compares their transcriptional profile with that of whole retinal tissue, as well as classical, intermediate and non-classical monocytes. In addition, human retinal microglia are compared to murine retinal microglia, isolated from at least two-years old Cx3cr1GFP/+ mice, as well as human brain microglia obtained from the literature. Several overexpressed genes were identified in retinal microglia when compared to whole retinal tissue, as well as classical, intermediate, and non-classical monocytes, among them IL1B, C2, C3, TREM2, P2RY12 and SPP1. In relation to whole retina sequencing, several risk genes, such as APOE and TGBR1, as well as PLXDC2 and ARHGAP22 associated with age-related macular degeneration (AMD) and diabetic retinopathy (DRP), were preferentially expressed in retinal microglia, indicating their potential pathophysiological involvement. The top expressed genes exhibited a strong consistency between retinal and brain microglia, among them CD74, SPP1, ACTB, FTL and C3. There was a high degree of similarity between human and murine retinal microglia, although there were several species-specific genes, revealing for which genes mice are suitable models for human retinal microglia. This study provides detailed insights into the molecular profile of human retinal microglia and indicate a high similarity to brain microglia. It advances our under-standing about their role in human retinal disease, such as AMD and DRP. The similarities and differences between human and mice will facilitate the transferability of knowledge between both species.
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Project description:Microglia are specialised brain-resident macrophages that arise from primitive macrophages colonising the embryonic brain. Microglia contribute to multiple aspects of brain development, but their precise roles in early human brain remain poorly understood due to limited access to relevant tissues. The generation of brain organoids from induced human pluripotent stem cells (iPSC) recapitulates some key features of human embryonic brain development, but current approaches do not incorporate microglia and thus are lacking. Here, we generated microgliasufficient brain organoids by co-culturing brain organoids with primitive-like macrophages generated from the same human iPSC (iMac). In organoid co-cultures, iMac differentiated into cells with microglia-like phenotypes and functions (iMicro), and modulated neuronal progenitor cell (NPC) differentiation, limiting NPC proliferation and promoting axonogenesis. Mechanistically, iMicro contained high levels of PLIN2+ lipid droplets that exported cholesterol and its esters which weretaken up by NPC in the organoids. We also detected PLIN2+ lipid droplet-loaded microglia in mouse and human embryonic brain. Overall, our approach significantly advances current human brain organoid approaches by incorporating microglial
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Project description:Microglia are specialised brain-resident macrophages that arise from primitive macrophages colonising the embryonic brain. Microglia contribute to multiple aspects of brain development, but their precise roles in early human brain remain poorly understood due to limited access to relevant tissues. The generation of brain organoids from induced human pluripotent stem cells (iPSC) recapitulates some key features of human embryonic brain development, but current approaches do not incorporate microglia and thus are lacking. Here, we generated microgliasufficient brain organoids by co-culturing brain organoids with primitive-like macrophages generated from the same human iPSC (iMac). In organoid co-cultures, iMac differentiated into cells with microglia-like phenotypes and functions (iMicro), and modulated neuronal progenitor cell (NPC) differentiation, limiting NPC proliferation and promoting axonogenesis. Mechanistically, iMicro contained high levels of PLIN2+ lipid droplets that exported cholesterol and its esters which weretaken up by NPC in the organoids. We also detected PLIN2+ lipid droplet-loaded microglia in mouse and human embryonic brain. Overall, our approach significantly advances current human brain organoid approaches by incorporating microglial
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Project description:The apolipoprotein E4 (APOE4) allele is associated with an increased risk of Alzheimer’s disease and a decreased risk of glaucoma, but the underlying mechanisms remain poorly understood. Here we find that in two mouse glaucoma models and in human glaucomatous retinas, microglia transition to a neurodegenerative (MGnD) phenotype characterized by upregulation of Apoe and Lgals3 (Galectin-3). Mice in which Apoe was targeted in microglia or carrying the human APOE4 allele were protected from retinal ganglion cell (RGC) loss despite elevated intraocular pressure (IOP). Similar to Apoe–/– retinal microglia, APOE4 microglia did not upregulate MGnD genes, including Lgals3, following IOP elevation. Genetic and pharmacologic targeting of Galectin-3 ameliorated RGC degeneration, and Galectin-3 expression was attenuated in human APOE4 glaucoma samples. These results demonstrate that impaired activation of APOE4 microglia is protective in glaucoma, and that the APOE-Galectin-3 signaling pathway can be targeted to treat this blinding disease.
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