Transcriptomic profiling of retinal cells reveals a subpopulation of microglia/macrophages expressing Rbpms and Spp1 markers of retinal ganglion cells (RGCs) that confound identification of RGCs.

Analysis of retinal ganglion cells (RGCs) by scRNA-seq is emerging as a state-of-the-art method for studying RGC biology and subtypes, as well as for studying the mechanisms of neuroprotection and axon regeneration in the central nervous system (CNS). Rbpms has been established as a pan-RGC marker, and Spp1 has been established as an αRGC type marker. Here, we analyzed by scRNA-seq retinal microglia and macrophages, and found Rbpms+ and Spp1+ subpopulations of retinal microglia/macrophages, which pose a potential pitfall in scRNA-seq studies involving RGCs. We performed comparative analysis of cellular identity of the presumed RGC cells isolated in recent scRNA-seq studies, and found that Rbpms+ and Spp1+ microglia/macrophages confounded identification of RGCs. We also provide solutions for circumventing this potential pitfall in scRNA-seq studies, by including in RGC and αRGC selection criteria other pan-RGC and αRGC markers.