Single-cell transcriptome comparison of myeloid cells’ in the retina in response to transplantation of human stem-cell derived neurons and to ocular damage reveals the reversibility of the microglia activation.

RGC loss is permanent given that these cells cannot regenerate in mammals. Neuroregenerative strategies for managing optic neuropathies have mainly focused on restoring the lost RGC cell population in a glaucomatous retina. This treatment approach not only aims to halt disease progression, but also to restore the lost vision by replacing the damaged/lost RGCs through RGC transplantation. Significant strides have been made in this field of RGC transplantation and regeneration. As a first step of RGC transplantation, several protocols now exist to differentiate cells into primary RGCs, and several sources of donor RGC have been established including human ES and iPSC-derived RGCs. To achieve functional regeneration, the transplanted RGCs must survive, integrate, and extend their axons and establish connections within the retina and the brain. Of the many factors influencing initial donor RGCs survival, the innate immunity [ref] and primarily microglia reactivity is of particular interest. In ocular models of glaucoma, microglia have been heavily implicated in driving disease progression, promoting a pro-inflammatory environment. In some instances, activated microglia have been directly implicated in RGC loss by mediating phagocytosis of the damaged or injured RGCs. In line with this, our recent work, shows that activated host microglia are detrimental to donor RGCs, presumably via phagocytosis of the donor RGCs. We observed that pre-treatment of donor RGCs with FasL and annexin V to block the exposed phosphatidylserine residues of the stressed donor RGCs prior to transplantation reduced activation of host microglia, and better RGC survival rates post transplantation. Studies like these and others underscore the importance of modulating host microglia reactivity for better transplantation outcomes. To further improve donor RGC survival post transplantation, a better understanding of the host myeloid cell reactivity following donor RGC transplantation is paramount. We and others have previously shown the importance of modulating the retinal microenvironment to promote RGC survival. In the current study, we profiled the transcriptome of the host myeloid cell population (CX3CR1-GFP) using a single cell RNA sequencing approach, to delineate the myeloid cell population most relevant for donor RGC survival post transplantation. A focused analysis on the microglia cell population was performed, given their established role in glaucoma disease progression. In addition to profiling the expression pattern of the known classic microglia disease associated signatures such as ApoE, Spp1, and Lgals, we identified several other genes associated with, and or driving microglia activation, including Cybb, Atf3, Aurka and Kif22. Given the known function of these genes in other disease contexts, we believe that these genes are important for modulating donor RGC survival post transplantation, thus warrant further investigation in this context. Taken together, with reference to our established adult mouse retina cell atlas, we present a comprehensive analysis of the host myeloid cell population, specifically focusing on microglia reactivity status, at a single cell resolution level, providing insights into the regulatory mechanisms that may be important for donor RGC survival and integration into glaucomatous retina post transplantation.