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

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Jinlun Bai , David S. Koos , Kayla Stepanian , Zachary Fouladian , Dominic WH Shayler , Jennifer G. Aparicio , Scott E. Fraser , Rex Moats , David Cobrinik ·

YOU? · · 2023 · Open Access · · DOI: https://doi.org/10.1101/2023.02.28.530518

Fluorescent reporter pluripotent stem cell (PSC) 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 allele robustly and exclusively labeled both immature and mature cones starting at culture day 34. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of morphological maturation of individual cones for >18 weeks and revealed inner segment accumulation of mitochondria and growth at 12.2 cubic microns 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.

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Organoid

Green Fluorescent Protein

Concepts

Induced pluripotent stem cell Organoid Green fluorescent protein Biology Cell biology Live cell imaging Retinal Growth cone HEK 293 cells Embryonic stem cell Cell culture Cell Genetics Gene Biochemistry Axon

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Type: preprint
Language: en
Landing Page: https://doi.org/10.1101/2023.02.28.530518
PDF: https://www.biorxiv.org/content/biorxiv/early/2023/03/01/2023.02.28.530518.full.pdf
OA Status: green
References: 30
Related Works: 10
OpenAlex ID: https://openalex.org/W4323049085

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DOI: https://doi.org/10.1101/2023.02.28.530518

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

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Type: preprint

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Language: en

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Publication year: 2023

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Publication date: 2023-03-01

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Authors: Jinlun Bai, David S. Koos, Kayla Stepanian, Zachary Fouladian, Dominic WH Shayler, Jennifer G. Aparicio, Scott E. Fraser, Rex Moats, David Cobrinik

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Landing page: https://doi.org/10.1101/2023.02.28.530518

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PDF URL: https://www.biorxiv.org/content/biorxiv/early/2023/03/01/2023.02.28.530518.full.pdf

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Concepts: Induced pluripotent stem cell, Organoid, Green fluorescent protein, Biology, Cell biology, Live cell imaging, Retinal, Growth cone, HEK 293 cells, Embryonic stem cell, Cell culture, Cell, Genetics, Gene, Biochemistry, Axon

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