Deciphering the Molecular Signature of Human Hyalocytes in Relation to Other Innate Immune Cell Populations. 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. Overall design: 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.