June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
A single-cell roadmap of photoreceptor development in human ESC models
Author Affiliations & Notes
  • Xiying Mao
    Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, China
  • Yuhua Xiao
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, China
  • Peiyao Mao
    Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, China
  • Songtao Yuan
    Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, China
  • Qinghuai Liu
    Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, China
  • youjin hu
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, China
  • Footnotes
    Commercial Relationships   Xiying Mao, None; Yuhua Xiao, None; Peiyao Mao, None; Songtao Yuan, None; Qinghuai Liu, None; youjin hu, None
  • Footnotes
    Support  National Key R&D Program of China (2017YFA0104101); National Natural Science Foundation of China (81970821)
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 2514. doi:
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      Xiying Mao, Yuhua Xiao, Peiyao Mao, Songtao Yuan, Qinghuai Liu, youjin hu; A single-cell roadmap of photoreceptor development in human ESC models. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2514.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : During retinal development, photoreceptors are produced by retinal progenitor cells and then committed to cone and rod subtypes in a stereotypical order. However, the mechanism that controls photoreceptor specification and its subsequent fate bifurcation remain elusive. Furthermore, the heterogeneity of photoreceptor lineage during development impeded our seeking for molecular markers of specific developmental stage which is vital for cell-based therapeutic application. Therefore, the goal of the study is to investigate molecular characteristics and mechanism during photoreceptor development at the single-cell resolution.

Methods : We first engineered H9 hESC reporter line using CRISPR/CAS9 gene-editing technology to fuse EGFP to endogenous BLIMP1. This engineered cell line was used to generate 3D retinal organoid. Following EGFP-based cell sorting, single-cell RNA-sequencing was conducted via 10x Genomics Chromium system. Systematic approaches including single-cell pseudotime analysis, single-cell trajectory reconstruction, and gene regulatory network analysis (SCENIC) were performed.

Results : We profiled a total of 10455 EGFP+ cells isolated from retinal organoids at three time points (Day 45, 80 and 120). Unbiased clustering detected 8 biological clusters, covering most cell states during photoreceptor genesis. Novel transcriptional regulators and specific surface markers for each developmental stage were identified and validated by immunostaining. Additionally, we deconstructed the trajectory of photoreceptor development, revealing the branchpoint of cone and rod lineage. Though identifying the differential molecular events between cone and rod branches, we found key signaling pathways that might temporally regulate photoreceptor subtype specification in humans. Experimental manipulation of these pathways will be performed to examine their impact on cone/rod fate bifurcation.

Conclusions : By taking advantage of human embryonic stem cell-derived retinal organoid as a model of studying human retinal development, molecular continuum of photoreceptor development was dissected at the single-cell resolution. The identification of surface markers and mechanism that controls fate choice of cone versus rod fate will provide important guidance for the enrichment and direct differentiation of purified photoreceptor subtypes in cell replacement therapy to treat end-stage retinal degenerative disease.

This is a 2020 ARVO Annual Meeting abstract.

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