Episodic Live Imaging of Cone Photoreceptor Maturation in GNAT2-EGFP Retinal Organoids

Overview

Journal Dis Model Mech

Specialty General Medicine

Date 2023 Oct 30

PMID 37902188

Authors

Jinlun Bai

David S Koos

Kayla Stepanian

Zachary Fouladian

Dominic W H Shayler

Jennifer G Aparicio

Scott E Fraser

Rex A Moats

David Cobrinik

Affiliations

Soon will be listed here.

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.

Citing Articles

Non-canonical Wnt pathway expression in the developing mouse and human retina.

Campos R, Matsunaga K, Reid M, Fernandez G, Stepanian K, Bharathan S Exp Eye Res. 2024; 244:109947.

PMID: 38815793 PMC: 11179970. DOI: 10.1016/j.exer.2024.109947.

Classical and Innovative Evidence for Therapeutic Strategies in Retinal Dysfunctions.

Caruso L, Fields M, Rimondi E, Zauli G, Longo G, Marcuzzi A Int J Mol Sci. 2024; 25(4).

PMID: 38396799 PMC: 10889839. DOI: 10.3390/ijms25042124.

References

KELLEY M, Turner J, Reh T . Retinoic acid promotes differentiation of photoreceptors in vitro. Development. 1994; 120(8):2091-102. DOI: 10.1242/dev.120.8.2091. View

Ronning K, Peinado Allina G, Miller E, Zawadzki R, Pugh Jr E, Herrmann R . Loss of cone function without degeneration in a novel Gnat2 knock-out mouse. Exp Eye Res. 2018; 171:111-118. PMC: 5987249. DOI: 10.1016/j.exer.2018.02.024. View

Browne A, Arnesano C, Harutyunyan N, Khuu T, Martinez J, Pollack H . Structural and Functional Characterization of Human Stem-Cell-Derived Retinal Organoids by Live Imaging. Invest Ophthalmol Vis Sci. 2017; 58(9):3311-3318. PMC: 5495152. DOI: 10.1167/iovs.16-20796. View

Kaewkhaw R, Kaya K, Brooks M, Homma K, Zou J, Chaitankar V . Transcriptome Dynamics of Developing Photoreceptors in Three-Dimensional Retina Cultures Recapitulates Temporal Sequence of Human Cone and Rod Differentiation Revealing Cell Surface Markers and Gene Networks. Stem Cells. 2015; 33(12):3504-18. PMC: 4713319. DOI: 10.1002/stem.2122. View

Xu X, Singh H, Wang L, Qi D, Poulos B, Abramson D . Rb suppresses human cone-precursor-derived retinoblastoma tumours. Nature. 2014; 514(7522):385-8. PMC: 4232224. DOI: 10.1038/nature13813. View

Gagliardi G, Ben MBarek K, Chaffiol A, Slembrouck-Brec A, Conart J, Nanteau C . Characterization and Transplantation of CD73-Positive Photoreceptors Isolated from Human iPSC-Derived Retinal Organoids. Stem Cell Reports. 2018; 11(3):665-680. PMC: 6135113. DOI: 10.1016/j.stemcr.2018.07.005. View

Mustafi D, Engel A, Palczewski K . Structure of cone photoreceptors. Prog Retin Eye Res. 2009; 28(4):289-302. PMC: 2740621. DOI: 10.1016/j.preteyeres.2009.05.003. View

Wei X, Henke V, Strubing C, Brown E, Clapham D . Real-time imaging of nuclear permeation by EGFP in single intact cells. Biophys J. 2003; 84(2 Pt 1):1317-27. PMC: 1302708. DOI: 10.1016/S0006-3495(03)74947-9. View

Guan Y, Wang Y, Zheng D, Xie B, Xu P, Gao G . Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation. Front Cell Dev Biol. 2022; 10:870441. PMC: 9096726. DOI: 10.3389/fcell.2022.870441. View

10.

Lam P, Gutierrez C, Del Rio-Tsonis K, Robinson M . Generation of a Retina Reporter hiPSC Line to Label Progenitor, Ganglion, and Photoreceptor Cell Types. Transl Vis Sci Technol. 2020; 9(3):21. PMC: 7352077. DOI: 10.1167/tvst.9.3.21. View

11.

Tram N, Swindle-Reilly K . Rheological Properties and Age-Related Changes of the Human Vitreous Humor. Front Bioeng Biotechnol. 2019; 6:199. PMC: 6305337. DOI: 10.3389/fbioe.2018.00199. View

12.

Hendrickson A, Zhang C . Development of cone photoreceptors and their synapses in the human and monkey fovea. J Comp Neurol. 2017; 527(1):38-51. PMC: 6529189. DOI: 10.1002/cne.24170. View

13.

Sluch V, Chamling X, Liu M, Berlinicke C, Cheng J, Mitchell K . Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells. Stem Cells Transl Med. 2017; 6(11):1972-1986. PMC: 6430043. DOI: 10.1002/sctm.17-0059. View

14.

Morris T, Fong W, Ward M, Hu H, Fong S . Localization of upstream silencer elements involved in the expression of cone transducin alpha-subunit (GNAT2). Invest Ophthalmol Vis Sci. 1997; 38(1):196-206. View

15.

Phillips M, Capowski E, Petersen A, Jansen A, Barlow K, Edwards K . Generation of a rod-specific NRL reporter line in human pluripotent stem cells. Sci Rep. 2018; 8(1):2370. PMC: 5799252. DOI: 10.1038/s41598-018-20813-3. View

16.

Wahlin K, Maruotti J, Sripathi S, Ball J, Angueyra J, Kim C . Photoreceptor Outer Segment-like Structures in Long-Term 3D Retinas from Human Pluripotent Stem Cells. Sci Rep. 2017; 7(1):766. PMC: 5429674. DOI: 10.1038/s41598-017-00774-9. View

17.

Capowski E, Samimi K, Mayerl S, Phillips M, Pinilla I, Howden S . Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines. Development. 2018; 146(1). PMC: 6340149. DOI: 10.1242/dev.171686. View

18.

Singh H, Wang S, Stachelek K, Lee S, Reid M, Thornton M . Developmental stage-specific proliferation and retinoblastoma genesis in RB-deficient human but not mouse cone precursors. Proc Natl Acad Sci U S A. 2018; 115(40):E9391-E9400. PMC: 6176579. DOI: 10.1073/pnas.1808903115. View

19.

Hoshino A, Ratnapriya R, Brooks M, Chaitankar V, Wilken M, Zhang C . Molecular Anatomy of the Developing Human Retina. Dev Cell. 2017; 43(6):763-779.e4. PMC: 5776731. DOI: 10.1016/j.devcel.2017.10.029. View

20.

Aparicio J, Hopp H, Harutyunyan N, Stewart C, Cobrinik D, Borchert M . Aberrant gene expression yet undiminished retinal ganglion cell genesis in iPSC-derived models of optic nerve hypoplasia. Ophthalmic Genet. 2023; 45(1):1-15. PMC: 10841193. DOI: 10.1080/13816810.2023.2253902. View