Molecular taxonomy of human ocular outflow tissues defined by single-cell transcriptomics Gaurang Patela,1, Wen Furya,1, Hua Yanga, Maria Gomez-Caraballob, Yu Baia, Tao Yanga, Christina Adlera, Yi Weia, Min Nia, Heather Schmittb, Ying Hua, George Yancopoulosa, W. Daniel Stamerb,2, and Carmelo Romanoa,2 aRegeneron Pharmaceuticals, Inc., Tarrytown, NY 10591; and bDepartment of Ophthalmology, Duke University, Durham, NC 27710 Edited by Joel S. Schuman, New York University, New York, NY, and accepted by Editorial Board Member Jeremy Nathans April 16, 2020 (received for review January 31, 2020) The conventional outflow pathway is a complex tissue responsible refs. 7 and 8). Cells that populate the TM are all of neural crest in for maintaining intraocular pressure (IOP) homeostasis. The co- origin (13), displaying different morphologies depending upon their ordinated effort of multiple cells with differing responsibilities tissue location. Thus, TM cells in uveal/corneoscleral meshwork ensures healthy outflow function and IOP maintenance. Dysfunc- display endothelial and macrophage properties, maintaining patent tion of one or more resident cell types results in ocular hyperten- flow passageways by secreting antithrombotic molecules (14), sion and risk for glaucoma, a leading cause of blindness. In this phagocytosing cellular debris, neutralizing reactive oxygen species study, single-cell RNA sequencing was performed to generate a (15–17), and mediating immune function (18, 19), while the cells in comprehensive cell atlas of human conventional outflow tissues. the JCT region display fibroblast- and smooth muscle-like proper- We obtained expression profiles of 17,757 genes from 8,758 cells ties, playing an important role in the generation and control of from eight eyes of human donors representing the outflow cell outflow resistance (7). In response to various stimuli including transcriptome. Upon clustering analysis, 12 distinct cell types were mechanical cues (20–22), JCT cells continuously repair/remodel the identified, and region-specific expression of candidate genes was ECM (23) and maintain contractile tissue tone in conjunction with mapped in human tissues. Significantly, we identified two distinct the ciliary muscle. The importance of TM contractile tone in IOP expression patterns (myofibroblast- and fibroblast-like) from cells regulation has been recently exploited pharmacologically by a new located in the trabecular meshwork (TM), the primary structural class of glaucoma drugs, the rho-kinase inhibitors, which selectively component of the conventional outflow pathway. We also located relax the TM and decrease outflow resistance (24). Schwann cell and macrophage signatures in the TM. The second Studies involving the use of TM and SC cells in cell and organ CELL BIOLOGY primary component structure, Schlemm’s canal, displayed a unique culture have resulted in the identification of multiple drug targets combination of lymphatic/blood vascular gene expression. Other expression clusters corresponded to cells from neighboring tissues, Significance predominantly in the ciliary muscle/scleral spur, which together correspond to the uveoscleral outflow pathway. Importantly, the Ocular hypertension is the primary and only modifiable risk utility of our atlas was demonstrated by mapping glaucoma-relevant factor for glaucoma, the leading cause of irreversible blindness. genes to outflow cell clusters. Our study provides a comprehensive Intraocular pressure is regulated homeostatically by resistance molecular and cellular classification of conventional and unconven- to aqueous humor outflow through an architecturally complex tional outflow pathway structures responsible for IOP homeostasis. tissue, the conventional/trabecular pathway. In this study, we generated a comprehensive cell atlas of the human trabecular glaucoma | trabecular meshwork | outflow facility | single-cell meshwork and neighboring tissues using single-cell RNA se- transcriptomics | anterior chamber quencing. We identified 12 distinct cell types and mapped region-specific expression of candidate genes. The utility of our levated intraocular pressure (IOP) is a major causative risk atlas was demonstrated by mapping glaucoma-relevant genes Efactor for the development (1) and progression (2, 3) of most to conventional outflow cell types. Our study provides a com- forms of glaucoma. Efficacious lowering of IOP, whether ele- prehensive molecular and cellular classification of tissue struc- vated or not, slows glaucomatous disease progression (2, 4). Cur- tures responsible for intraocular pressure homeostasis in rent medical treatments lower IOP by three different mechanisms: health and dysregulation in disease. suppressing aqueous humor formation, or increasing its rate of outflow via the unconventional or conventional outflow pathways Author contributions: G.P., W.F., Y.H., G.Y., W.D.S., and C.R. designed research; G.P., W.F., (5, 6). While all three target tissues participate in aqueous humor H.Y., M.G.-C., H.S., and W.D.S. performed research; G.P., W.F., H.Y., M.G.-C., Y.B., T.Y., C.A., Y.W., M.N., Y.H., G.Y., W.D.S., and C.R. analyzed data; and G.P., W.F., Y.H., G.Y., dynamics, the conventional (trabecular) outflow pathway is re- W.D.S., and C.R. wrote the paper. sponsible for homeostatically regulating IOP by the coordinated Competing interest statement: G.P., W.F., H.Y., Y.B., T.Y., C.A., Y.W., M.N., Y.H., G.Y., and generation of outflow resistance involving cells that reside in the C.R. are employees and shareholders of Regeneron Pharmaceuticals, although the man- trabecular meshwork (TM) and Schlemm’s canal (SC) (7–10). uscript’s subject matter does not have any relationship to any products or services of this Importantly, dysfunction in the regulation of conventional outflow corporation. resistance results in elevated IOP in glaucoma patients (11, 12). This article is a PNAS Direct Submission. J.S.S. is a guest editor invited by the The TM is an avascular, complex connective tissue located at Editorial Board. the iridocorneal angle, bridging from Schwalbe’s line anteriorly This open access article is distributed under Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND). to the scleral spur/ciliary muscle posteriorly. Anatomically, the Data deposition: The raw data files from this study have been deposited in the Sequence TM is divided into three distinct tissue layers: the inner uveal Read Archive of the National Center for Biotechnology Information (accession no. meshwork, middle corneoscleral meshwork, and outer juxtaca- PRJNA616025). nalicular tissue (JCT, also known as cribriform tissue). Function- 1G.P. and W.F. contributed equally to this work. ing as a biological filter, the uveoscleral and corneoscleral 2To whom correspondence may be addressed. Email: [email protected] or carmelo. meshwork consists of collagen and elastin lamellae/plates covered [email protected]. by TM cells. In contrast, the JCT is composed of TM cells em- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ bedded in the extracellular matrix (ECM) of a loose connective doi:10.1073/pnas.2001896117/-/DCSupplemental. tissue, directly interfacing with the inner wall of SC (reviewed in www.pnas.org/cgi/doi/10.1073/pnas.2001896117 PNAS Latest Articles | 1of12 Downloaded by guest on September 29, 2021 Fig. 1. Identification of human outflow cell types. (A)At-distributed stochastic neighbor embedding (tSNE) visualization of outflow tissue transcriptome heterogeneity of 8,758 cells. Twelve distinct cell clusters were identified in the outflow tract, and cells are colored and labeled by cluster assignments. (B) Dot plot of gene expression combinations that uniquely identified each of the cell clusters. Quality control metrics are described in Materials and Methods.The size of each circle is proportional to the percentage of cells expressing the gene (marker) and its intensity depicts the normalized average transcript count of all cells within the cluster. (C) Hierarchical clustering of cell types, showing similarity or dissimilarity among cell types. 2of12 | www.pnas.org/cgi/doi/10.1073/pnas.2001896117 Patel et al. Downloaded by guest on September 29, 2021 that are in various stages of clinical development, including rho- isolated TM tissue from eight different human donor eyes using kinase inhibitors (25). However, due to their multiple responsi- a blunt dissection (SI Appendix, Fig. S1) and subjected the dis- bilities and unique environment, specific markers have not been sected tissue to enzymatic digestion to prepare a single-cell sus- identified for conventional outflow cells. Instead, cell morphology, pension. As reflected in SI Appendix, Fig. S1, there was a portion growth characteristics, behavior, dissection technique, and a panel of the ciliary muscle and scleral spur region included in the dis- of protein markers are used for their identification and charac- section. Single-cell suspensions were checked for cell viability, with terization. A typical panel of markers used to identify TM cells cells from four donor pairs (eight samples) included in subsequent includes chitinase-3 like-1 (CHI3L1), matrix GLA protein (MGP), droplet-based scRNAseq using the 10x Genomics platform (https:// aquaporin-1 (25–28), and glucocorticoid-induced up-regulation of www.10xgenomics.com/). Unique molecular identifiers (UMIs) are myocilin (MYOC) (29). Several transcriptomic and proteomic molecular tags that are used to detect