Single-cell transcriptome of retinal myeloid cells in response to transplantation of human neurons reveals reversibility of microglial activation.

The host retinal microglia and macrophage activation remains a major challenge for the integration of donor neurons following transplantation. Previously, we and others have shown that it is possible to increase donor retinal ganglion cell (RGC) survival by inhibiting the microglia-RGC interaction with Annexin V or through reprogramming microglia with the soluble Fas ligand. However, the exact mechanisms of the microglia/macrophage activation and their heterogeneity following transplantation remain unknown. To address this question, the donor RGC were differentiated from Brn3b-Tdtomato-Thy1.2 human embryonic stem cells using a 3D protocol, followed by dissociation and RGC purification. RGC were delivered subretinally (1.5×10 viable cells/eye) into 3-6-month-old knock-in mice. Three days after transplantation retinas were dissociated into single-cell suspension and GFP-positive myeloid cells isolated using FACS. Of the sorted cells, up to 10,000 viable cells per sample were used for single-cell RNA library preparation and sequenced using the 10X Genomics Chromium platform. In addition, several retinas were fixed and stained for donor RGC (mCherry) and host microglia/macrophages (Iba1). RNA Velocity was used to reconstruct the myeloid cell population and activation trajectory from scRNAseq data. We observed continuous bi-directional transition of microglia/macrophages from a homeostatic to an activated state. We also observed that the response to the transplant falls into the classic disease-associated-microglia (DAM) activation paradigm with a decrease in expression of the homeostatic gene and an increase in expression of disease-associated genes including and . Our findings show that the host retinal myeloid cell population undergoes activation upon transplantation of stem-cell derived donor RGC, with a molecular profile of the activated cells similar to that of activated myeloid cells associated with neurodegenerative diseases of the brain and the eye. Advanced integrated transcriptomic analysis shows separate activated-to-homeostatic and homeostatic-to-activated trajectories suggesting the reversibility of this process.