Abstract
Purpose :
Members of the transforming growth factor β (TGFβ) superfamily play a key role in development, functioning in stem cell maintenance and organ morphogenesis through its role in the regulation of pluripotency. Aberrant TGFβ signalling has been implicated in many inflammatory retinal conditions although the underlying pathology has not yet been well-characterised.
Herein, we propose an organoid model of retinal inflammation, by stimulating retinal organoids with the inflammatory cytokine transforming growth factor β II (TGFβII) and subsequently performing single cell RNA sequencing (scRNAseq) to interrogate its effects on the differentiation of retinal cells.
Methods :
Retinal organoids (ROs) were differentiated from induced pluripotent stem cells (iPSCs) using an optimised protocol over a period of 76 days. ROs were stimulated with 10ng/μL TGFβII for a duration of 7 days before undergoing dissociation and subjected to scRNAseq (N=6 organoids per condition, N=3 independent differentiations). Datasets were pre-processed and filtered using the Seurat v4.0 package prior to normalisation and dimensional reduction with principal component analysis for further downstream analysis.
Results :
Differential gene expression analysis revealed global downregulation of genes associated with immune signalling (PLCG2, avg_log2FC = 1.774), cell migration (ROBO2, avg_log2FC = 0.567) and synaptic transmission (LRRTM4, avg_log2FC = 0.564) in the TGFβII-treated group. Global upregulation of genes such as IGFBP5, associated with (avg_log2FC = -0.944) and the pro-angiogenic factor, MDK (avg_log2FC = -0.881) was also noted. Cluster by cluster analysis revealed different responses to TGFβII treatment depending on cell type, with photoreceptor precursors showing downregulation in NEUROD4 (avg_log2FC = 0.599), PDE1C (avg_log2FC = 0.493) and PRDM1 (avg_log2FC = 0.4956) suggesting that TGFβII treatment may be affecting photoreceptor polarisation.
Conclusions :
Our results indicate that TGFβII treatment is indeed affecting the differentiation of various retinal cell types, with the most prominently affected being the developing photoreceptor cells. Further analysis to elucidate the mechanism by which this is occuring may reveal new pathways which could be exploited for therapeutic benefit.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.