Harnessing trabecular meshwork cells for the molecular profiling of primary open-angle glaucoma

Background: Primary open-angle glaucoma (POAG) is the leading cause of irreversible blindness worldwide. Intraocular pressure (IOP) the only known modifiable risk factor for this disease. In POAG, elevated IOP leads to progressive loss of retinal ganglion cells, with IOP being primarily modulated by trabecular meshwork cells (TMCs) in the iridocorneal angle. The precise molecular drivers for the differentiation or dysfunction of TMCs is poorly understood. A number of transcription factors have been implicated in development of the ocular anterior segment, and recently a large genome-wide association study identified 53 loci associated with variation in IOP. Aim: The overriding aims of this thesis were: 1. To determine whether dental pulp-Mesenchymal stem cells (DP-MSCs) could be induced to exhibit the phenotypic properties of human TMCs through the exposure to growth factors involved in development of the anterior segment; 2. To investigate the morphological and transcriptome profiles created by Cell Painting and scRNA-seq in TMCs, where genes implicated in IOP variation have been knocked out by CRISPR. Methods: DP-MSCs were treated with retinoic acid (RA), TGF-˜í‚⧠super-family members transforming growth factor-˜í‚â§2 (TGF-˜í‚â§2) and bone morphogenetic protein 4 (BMP4) for 5 days. The expressions of TMCs markers were investigated by qRT-PCR, functional TM cell characteristics were profiled by a collagen contraction assay, dexamethasone-induced myocilin secretion and through phagocytosis assessment. To investigate genes associated with IOP, a clustered regularly interspaced short palindromic repeats (CRISPR) knockout screen was performed by transfecting the TMCs with lentivirus carrying the single-guide RNAs (sgRNAs) targeting 62 genes across 53 loci, together with 5 human non-targeting controls, in arrayed format. Cells were then seeded to the image plates by flow cytometry for high-throughput morphological profiling assay, or pooled for single-cell RNA sequencing (scRNA-seq). Results: After 5 days of treatment with the combination of the three transcription factors, RA, TGF-˜í‚â§2 and BMP4 in the DP-MSCs, the TMCs marker MYOC were significantly upregulated, and the secreted myocilin was also upregulated determined by ELISA; however, the expression of MYOC was not increased or induced following dexamethasone exposure and the contractility of collagen gel was also not observed. A total of 210, 234 cells were individually segmented and image-based profiling performed for CRISPR/Cas edited TM cells. A total of 910 morphological features were extracted for each of the gene knockout perturbations. Cells clustered into 2 major groups via unstructured hierarchical clustering. Significant features of the clusters were extracted related to the granularity of the golgi apparatus and mitochondrial. For transcriptional profiling a total of 25,879 single cells were demultiplexed and had their corresponding sgRNA identified. A total of 240 differentially expressed genes (DEGs) were identified and expression profiles with these genes. The profiles were also clustered into 2 groups via hierarchical clustering, the DEGs related to the difference of the 2 clusters are involved in the interferon alpha/beta signaling. Conclusion: Although DP-MSCs express markers of trabecular meshwork cells following the exposure of a combination of transcription factors, they were found not to acquire morphological profiles characteristic of trabecular meshwork function. High throughput analysis of cellular structure and function through cellpainting and scRNA-seq assays enabled the direct study of genetic perturbations at the single cell resolution. This work provides a framework for investigating the role of genes involved in the pathogenesis of glaucoma in both genetic and morphology.