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Jie Cheng, Melissa Liu, Aniruddha M. Kaushik, Xiaoli Chang, Yukan Duan, Liben Chen, Jeff (Tza-Huei) Wang, Cynthia Berlinicke, Donald J Zack; Single-Cell Transcriptome Profiling of Human Stem Cell-Derived Retinal Ganglion Cells in a Dominant Optic Atrophy Model. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1988. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Degeneration of subtype-specific retinal ganglion cells (RGCs) occurs in patients with hereditary optic neuropathies such as Dominant Optic Atrophy (DOA) as well as in various animal models of optic nerve degeneration. While murine RGC subtypes are well-characterized, little is known about the molecular identities of human RGCs. To begin to address this, we performed single cell RNA-seq (scRNA-seq) on human stem cell-derived RGCs as well as on congenic RGCs from a DOA model harboring OPA1 mutations introduced by CRISPR-Cas9 genome editing.
scRNA-seq was performed on a homemade Drop-seq device. Briefly, RGCs differentiated from human stem cells through small molecule-directed differentiation were enzymatically dissociated into single cells, immuno-purified, and subjected to microfluidic encapsulation in oil droplets together with barcoded beads. mRNAs from single cells were captured by the beads and then reverse transcribed, amplified, fragmented, and indexed with Illumina NEXTERA XT primers and custom primers. The resulting single cell libraries were sequenced on the Illumina HiSeq platform. Sequence reads were aligned to the human genome using the Drop-seq pipelines on a computer cluster. Subsequent data analysis was performed using an R package, Seurat (v2.0), on a Windows PC.
t-distributed stochastic neighbor embedding (t-SNE) and clustering of scRNA-seq data from wild type and OPA1 mutant RGCs revealed 8 district clusters. While RGC markers POU4F2, ISL1, SPP1, NRN1, NHLH2, SNCG and POU6F2 were presented in these clusters, markers of other retinal cell types such as RCVRN, RPE65, PTGDS, GRM6, RLBP1 and LHX1 were absent. Members of the SoxC transcription factors (Sox4 and Sox11) which were recently implicated in RGC fate specification, axon guidance, and regeneration were also highly expressed.
In this study, we have performed transcriptional analysis of human stem cell-derived RGCs at the single cell level. Consistent with the pathology in patients with DOA, RGC fate specification of OPA1 mutants was not significantly changed. Further analysis comparing mutant and wild type RGCs under stress conditions could provide insights into mutant RGC degeneration as well as the strategies for regeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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