Single-cell transcriptomics reveals reversible microglia/macrophage activation after human retinal neuron transplantation into mouse retina

Host retinal microglia and macrophages can limit donor neuron survival and integration after transplantation, but the cellular mechanisms underlying this response remain unclear. In this study, we used human embryonic stem cell-derived retinal ganglion cells, generated from Brn3b-tdTomato-Thy1.2-derived retinal organoids, and transplanted them subretinally into healthy adult CX3CR1^GFP mice. Three days later, GFP-positive host myeloid cells were isolated by fluorescent activated cell sorting and profiled using 10× single-cell RNA sequencing, with immunohistochemistry validating spatial and molecular changes. Transcriptomic and RNA velocity analyses revealed a microglia/macrophage activation continuum, including homeostatic, transitory, proliferative, and activated states. Transplantation induced a disease-associated microglia-like program, marked by reduced Tmem119 and increased Apoe, Lgals3, Spp1, Cybb, and related activation genes. Integrated atlas comparisons linked this response to retinal and neurodegenerative disease-associated myeloid states. Importantly, separate homeostatic-to-activated and activated-to-homeostatic trajectories suggest that transplantation-induced host myeloid activation is dynamic and partially reversible, with implications for improving future retinal cell therapies.