Single-Cell RNA Sequencing of hESC-Derived 3D Retinal Organoids Reveals Novel Genes Regulating RPC Commitment in Early Human Retinogenesis

Xiying Mao; Qin An; Huiyu Xi; Xian-Jie Yang; Xiangmei Zhang; Songtao Yuan; Jinmei Wang; Youjin Hu; Qinghuai Liu; Guoping Fan

doi:10.1016/j.stemcr.2019.08.012

Single-Cell RNA Sequencing of hESC-Derived 3D Retinal Organoids Reveals Novel Genes Regulating RPC Commitment in Early Human Retinogenesis

Stem Cell Reports. 2019 Oct 8;13(4):747-760. doi: 10.1016/j.stemcr.2019.08.012. Epub 2019 Sep 19.

Authors

Xiying Mao¹, Qin An², Huiyu Xi³, Xian-Jie Yang⁴, Xiangmei Zhang⁴, Songtao Yuan¹, Jinmei Wang⁵, Youjin Hu⁶, Qinghuai Liu⁷, Guoping Fan⁸

Affiliations

¹ Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
² Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, 695 Charles Young Drive South, Los Angeles, CA 90095, USA.
³ Department of Ophthalmology, Xuzhou First People's Hospital of Xuzhou Medical University, Xuzhou Eye Research Institute, Xuzhou 221002, China.
⁴ Stein Eye Institute, Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
⁵ Institute of Regenerative Medicine and International Lab of Ocular Stem Cells at Shanghai East Hospital, School of Life Science &Technology, Tongji University, Shanghai 200092, China.
⁶ State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China. Electronic address: huyoujin@gzzoc.com.
⁷ Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. Electronic address: liuqh@njmu.edu.cn.
⁸ Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, 695 Charles Young Drive South, Los Angeles, CA 90095, USA. Electronic address: gfan@mednet.ucla.edu.

Abstract

The development of the mammalian retina is a complicated process involving the generation of distinct types of neurons from retinal progenitor cells (RPCs) in a spatiotemporal-specific manner. The progression of RPCs during retinogenesis includes RPC proliferation, cell-fate commitment, and specific neuronal differentiation. In this study, by performing single-cell RNA sequencing of cells isolated from human embryonic stem cell (hESC)-derived 3D retinal organoids, we successfully deconstructed the temporal progression of RPCs during early human retinogenesis. We identified two distinctive subtypes of RPCs with unique molecular profiles, namely multipotent RPCs and neurogenic RPCs. We found that genes related to the Notch and Wnt signaling pathways, as well as chromatin remodeling, were dynamically regulated during RPC commitment. Interestingly, our analysis identified that CCND1, a G₁-phase cell-cycle regulator, was coexpressed with ASCL1 in a cell-cycle-independent manner. Temporally controlled overexpression of CCND1 in retinal organoids demonstrated a role for CCND1 in promoting early retinal neurogenesis. Together, our results revealed critical pathways and novel genes in early retinogenesis of humans.

Keywords: commitment; early human retinal development; human embryonic stem cell; retina; retinal progenitor; single-cell RNA-seq.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Biomarkers
Fluorescent Antibody Technique
Gene Expression Profiling
Gene Expression Regulation, Developmental*
Gene Regulatory Networks
High-Throughput Nucleotide Sequencing
Human Embryonic Stem Cells / cytology*
Human Embryonic Stem Cells / metabolism*
Humans
Immunophenotyping
Organogenesis / genetics*
Organoids*
Retina / cytology*
Retina / metabolism*
Single-Cell Analysis
Tissue Culture Techniques

Substances

Biomarkers

Abstract

Publication types

MeSH terms

Substances

Grants and funding