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

Jinlun Bai; David S Koos; Kayla Stepanian; Zachary Fouladian; Dominic W H Shayler; Jennifer G Aparicio; Scott E Fraser; Rex A Moats; David Cobrinik

doi:10.1242/dmm.050193

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

Dis Model Mech. 2023 Nov 1;16(11):dmm050193. doi: 10.1242/dmm.050193. Epub 2023 Nov 21.

Authors

Jinlun Bai^{1

2

3}, David S Koos^{2

4

5}, Kayla Stepanian^{1

2}, Zachary Fouladian^{1

2

3}, Dominic W H Shayler^{1

2

3}, Jennifer G Aparicio^{1

2}, Scott E Fraser^{2

4

6

7}, Rex A Moats^{2

4

5

6}, David Cobrinik^{1

2

8

9

10}

Affiliations

¹ The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA.
² The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA.
³ Development, Stem Cell, and Regenerative Medicine Program, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
⁴ Translational Biomedical Imaging Laboratory, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA.
⁵ Department of Radiology, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA.
⁶ Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089, USA.
⁷ Translational Imaging Center, University of Southern California, Los Angeles, CA 90089, USA.
⁸ Department of Ophthalmology and Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
⁹ Department of Biochemistry and Molecular Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
¹⁰ Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

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.

Keywords: CRISPR; Cone photoreceptor; Fluorescent reporter; GNAT2; Live imaging; Retinal organoid; iPSC.

MeSH terms

Cell Differentiation
Humans
Induced Pluripotent Stem Cells*
Organoids
Retina / metabolism
Retinal Cone Photoreceptor Cells* / metabolism

Substances

enhanced green fluorescent protein

Abstract

MeSH terms

Substances

Grants and funding