Purchase this article with an account.
Joseph Collin, Rachel Queen, Carla Bernadette Mellough, Majlinda Lako; Using hESC-derived retinal organoids to investigate the transcriptional profile of emerging photoreceptors. Invest. Ophthalmol. Vis. Sci. 2018;59(9):570.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Irreversible loss of photoreceptors is a hallmark of outer retinal diseases, which cause a significant proportion of irreversible blinding conditions. The ability to generate retinal organoids from human embryonic stem cells (hESCs) represents an opportunity to gain greater insight into the biology and potential of these lab-generated photoreceptors. The purpose of this study was to establish the feasibility of single cell RNA-sequencing (RNA-Seq) of hESC-derived photoreceptors during their differentiation in vitro.
Retinal organoids differentiated from a cone-rod homeobox (CRX)-green fluorescence protein (GFP) engineered hESC line for 90 days were dissociated and fluorescence-activated cell sorted into GFP-positive and negative populations. RNA-Seq was then performed on both populations. In addition up to 96 GFP positive cells were captured using the C1 Single-Cell mRNA Seq chip (Fluidigm) for single cell RNA-Seq.
The single cell isolation resulted in the capture of 90 cells, which were then subject to RNA-Seq and bioinformatic analysis. After filtering, 69 cells and 14887 genes passed quality control, which led to the acquisition of 200,000-500,000 aligned reads and the detection of 2000-8000 genes per cell. Unsupervised clustering using the SC3 R package was performed leading to the identification of 2 clusters. QuSAGE was used to identify sets of genes which were significantly differentially expressed between the two clusters. Ontology enrichment analysis showed that the larger cluster (50 cells) had a gene expression profile that was most similar to the transcriptional profile of cone and rod photoreceptors recently reported in Cell. 2015;161:1202-1214. These results indicated a largely homogenous population of hESC-derived CRX-GFP cells that were similar to murine adult photoreceptors.
The ability to generate a transcriptional profile of individual cells from a purified population of retinal organoid-derived CRX-positive cells allows insight into the heterogeneity and identity of these cells as they arise in vitro. Pathway analysis revealed terms such as neural retinal development and the cells showed the expression of known markers of photoreceptor precursors. Therefore this approach has the potential for detailed analysis of retinal organoids for the modelling of development and disease, drug discovery and cell replacement therapies.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
This PDF is available to Subscribers Only