Deciphering the Molecular Signature of Human Hyalocytes in Relation to Other Innate Immune Cell Populations

Purpose: The present study compares the transcriptional profiles of human hyalo-cytes, retinal microglia as well as classical, intermediate and non-classical mono-cytes in a pairwise analysis. Methods: Using fluorescence-activated cell sorting, hyalocytes and retinal microglia were isolated from the vitreous or retinal tissue of enucleated eyes due to melano-ma of the iris, ciliary body or choroid. In addition, classical, intermediate and non-classical monocytes were isolated from whole blood. RNA sequencing was per-formed and the transcriptional profiles of all five immune cell populations were ana-lyzed using bioinformatic analysis. Results: Pairwise analysis indicated distinct differences between hyalocytes and all three subsets of monocytes, whereas a high degree of similarity was apparent when compared to retinal microglia, with comparable expression levels of established MG markers such asTREM2, P2RY12 and TMEM119. Cluster analysis based on signa-ture genes of yolk sac-derived cells as well as hematopoietic stem and progenitor cells indicated that in contrast to monocytes, human hyalocytes and retinal microglia share a common origin in the yolk sac. Among the top expressed genes in hyalo-cytes, SPP1, CD74 and C3 were significantly upregulated when compared to mon-ocytes. Despite the high level of similarity, genes such as TNF, CCL3 and CXCL8 were significantly upregulated in hyalocytes when compared to retinal microglia. In addition, ten hyalocyte-specific genes were identified, among them FOS, DUSP1 and EGR2. Conclusions: This study provides detailed insights into the molecular profile of iso-lated human hyalocytes and, for the first time, human retinal microglia. The expres-sion profile of hyalocytes is similar to that of retinal microglia and indicates that hy-alocytes are, contrary to the prevailing notion, yolk sac-derived cells. The hyalocyte-specific markers identified in this study will contribute in the future to deciphering the different roles of this fascinating cell population in health and diseases of the vitre-oretinal interface. RNA sequencing of human hyalocytes (n=5), retinal microglia (n=5) as well as classical (n=6), intermediate (n=5) and non-classical (n=5) monocytes.