Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids

Author:

Bai Jinlun1234,Koos David S.356,Stepanian Kayla123,Fouladian Zachary1234,Shayler Dominic W. H.1234,Aparicio Jennifer G.123,Fraser Scott E.3578,Moats Rex A.3567,Cobrinik David12391011ORCID

Affiliation:

1. The Vision Center 1 , Department of Surgery , , Los Angeles, CA 90027 , USA

2. Children's Hospital Los Angeles 1 , Department of Surgery , , Los Angeles, CA 90027 , USA

3. The Saban Research Institute, Children's Hospital Los Angeles 2 , Los Angeles, CA 90027 , USA

4. Keck School of Medicine, University of Southern California 3 Development, Stem Cell, and Regenerative Medicine Program , , Los Angeles, CA 90033 , USA

5. Children's Hospital Los Angeles 4 Translational Biomedical Imaging Laboratory , , Los Angeles, CA 90027 , USA

6. Children's Hospital Los Angeles 5 Department of Radiology , , Los Angeles, CA 90027 , USA

7. University of Southern California 6 Department of Biomedical Engineering, Viterbi School of Engineering , , Los Angeles, CA 90089 , USA

8. Translational Imaging Center, University of Southern California 7 , Los Angeles, CA 90089 , USA

9. Keck School of Medicine, University of Southern California 8 Department of Ophthalmology and Roski Eye Institute , , Los Angeles, CA 90033 , USA

10. Keck School of Medicine, University of Southern California 9 Department of Biochemistry and Molecular Medicine , , Los Angeles, CA 90033 , USA

11. Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California 10 , Los Angeles, CA 90033 , USA

Abstract

ABSTRACT Fluorescent reporter pluripotent stem cell-derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus of guanine nucleotide-binding protein subunit alpha transducin 2 (GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP alleles robustly and exclusively labeled immature and mature cones. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of the morphological maturation of individual cones for >18 weeks and revealed inner segment accumulation of mitochondria and growth at 12.2 μm3 per day from day 126 to day 153. Immobilized GNAT2-EGFP cone reporter organoids provide a valuable tool for investigating human cone development and disease.

Funder

A. B. Reins Foundation

Knights Templar Eye Foundation

Neonatal Blindness Research Fund

Larry and Celia Moh Foundation

Research to Prevent Blindness

National Institutes of Health

Saban Research Institute, Children's Hospital Los Angeles

Publisher

The Company of Biologists

Subject

General Biochemistry, Genetics and Molecular Biology,Immunology and Microbiology (miscellaneous),Medicine (miscellaneous),Neuroscience (miscellaneous)

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