Human microglia impact neuronal development in retinal organoids

Prenatal immune challenges pose significant risks to human embryonic brain and eye development. However, we still lack knowledge about the safe usage of anti-inflammatory drugs during pregnancy. Human induced pluripotent stem cell (hIPSC)-derived brain organoid models provide a unique opportunity to investigate neuronal development and have started to explore functional consequences upon viral infection. However, brain organoids usually lack microglia, the brain-resident immune cells. They are present in the early human embryonic brain and actively participate in neuronal circuit development. At the same time, microglia are known for their immune-sensing properties and will influence viral-mediated effects. In my thesis, I was interested to study the multifunctional role of human microglia during retinal development. In chapter 1, I characterize the innate occurrence of IBA1+-microglia-like cells within the retinal organoid differentiation (Bartalska et al., 2022). Therefore, we differentiate hIPSC using an unguided retinal organoid differentiation protocol and observe the presence of IBA1+-microglia-like cells alongside retinal cups between week 3 and 4 in 2.5D culture. However, instead of infiltrating the neuroectodermal sides, they enrich within non-pigmented, 3D-cystic compartments that develop in low numbers parallel to 3D-retinal organoids. To enrich for IBA1+-microglia precursors (preMG), we guided the differentiation with a low-dosed BMP4 application, which prevents retinal cup development and enhances microglia and 3D-cysts formation. We characterize the differentiated preMG for their microglia-like identity and validated their functionality. In parallel, mass spectrometry identifies the 3D-cysts to express mesenchymal and epithelial markers. We confirm that comparable 3D-cysts are also the preferential environment for IBA1+-microglia-like cells within the unguided retinal organoid differentiation. In chapter 2, I investigate how microglia influence retinal development and whether they contribute to viral-mediated consequences (Schmied et al., 2025). Here, we assemble preMG, which we have characterized in chapter 1, into 3D-retinal organoids. Once the outer plexiform layer forms, microglia-like cells (iMG) populate them and interact with retinal cell types. However, at this developmental stage, the ganglion cell number decreases in 3D-retinal organoids. Thus, we adapted the model into 2D which promotes their survival. Integrated iMG engulf ganglion cells and control their cell number. In parallel, we apply the immunostimulant POLY(I:C) to mimic a fetal viral infection. Although POLY(I:C) stimulation affects iMG phenotype, it does not influence their interaction with ganglion cells. Furthermore, iMG presence significantly contributes to the supernatant’s inflammatory secretome and increases retinal cell proliferation. Simultaneous exposure to the non-steroidal anti-inflammatory drug (NSAID) ibuprofen dampens POLY(I:C)-mediated consequences of the iMG phenotype and ameliorates cell proliferation. Remarkably, while POLY(I:C) disrupts neuronal calcium dynamics independent of iMG presence, ibuprofen rescues this effect only in the presence of iMG. Mechanistically, ibuprofen blocks the enzymes cyclooxygenase 1 and 2 (COX1/ PTGS1 and COX2/ PTGS2) simultaneously, from which iMG predominantly express COX1. Selective inhibition of COX1 does not restore the calcium peak amplitude upon POLY(I:C) stimulation, indicating ibuprofen’s effect depends on the presence and interplay of both, COX1 and COX2. In summary, we characterized the 3D-retinal organoid model for the occurrence of IBA1+-microglia like cells. As the innately developing IBA1+-cells enrich in mesenchymal over retinal structures, we optimized a protocol to differentiate IBA1+-microglia precursors. By combining these two models we generate microglia-assembled retinal organoids. Our results underscore the importance of microglia during neurodevelopment, in the context of prenatal immune challenges and provide insight into the mechanisms by which ibuprofen exerts its protective effects during embryonic development.