Generation of a cone photoreceptor-specific GNGT2 reporter line in human pluripotent stem cells
Generation of a cone photoreceptor-specific GNGT2 reporter line in human pluripotent stem cells
Fluorescent reporter lines generated in human pluripotent stem cells are a highly useful tool to track, isolate, and analyze cell types and lineages in live cultures. Here, we generate the first human cone photoreceptor reporter cell line by CRISPR/Cas9 genome editing of a human embryonic stem cell (hESC) line to tag both alleles of the Guanine nucleotide-binding protein subunit gamma-T2 (GNGT2) gene with a mCherry reporter cassette. Three-dimensional optic vesicle-like structures were produced to verify reporter fidelity and track cones throughout their development in culture. The GNGT2-T2A-mCherry hESC line faithfully and robustly labels GNGT2-expressing cones throughout the entirety of their differentiation in vitro, recapitulating normal fetal expression of this gene. Our observations indicate that human cones undergo significant migratory activity during the course of differentiation in vitro. Consistent with this, our analysis of human fetal retinae from different stages of development finds positional differences of the cone population depending on their state of maturation. This novel reporter line will provide a useful tool for investigating human cone development and disease.
cone photoreceptor, GNGT2, optic vesicles, retinal organoids, stem cells
190-203
Nazlamova, Liliya
0cc21013-aeeb-4eef-af56-31f6fa0766fd
Cassidy, Emma Jane
5599a9bb-552e-446f-8889-961b901bea1e
Sowden, Jane C.
6f414289-9a33-42ad-a3cd-2fa828b7ac27
Lotery, Andrew
5ecc2d2d-d0b4-468f-ad2c-df7156f8e514
Lakowski, Jörn
1856e739-982a-412a-87c7-abf1610f5384
26 February 2022
Nazlamova, Liliya
0cc21013-aeeb-4eef-af56-31f6fa0766fd
Cassidy, Emma Jane
5599a9bb-552e-446f-8889-961b901bea1e
Sowden, Jane C.
6f414289-9a33-42ad-a3cd-2fa828b7ac27
Lotery, Andrew
5ecc2d2d-d0b4-468f-ad2c-df7156f8e514
Lakowski, Jörn
1856e739-982a-412a-87c7-abf1610f5384
Nazlamova, Liliya, Cassidy, Emma Jane, Sowden, Jane C., Lotery, Andrew and Lakowski, Jörn
(2022)
Generation of a cone photoreceptor-specific GNGT2 reporter line in human pluripotent stem cells.
Stem cells (Dayton, Ohio), 40 (2), .
(doi:10.1093/stmcls/sxab015).
Abstract
Fluorescent reporter lines generated in human pluripotent stem cells are a highly useful tool to track, isolate, and analyze cell types and lineages in live cultures. Here, we generate the first human cone photoreceptor reporter cell line by CRISPR/Cas9 genome editing of a human embryonic stem cell (hESC) line to tag both alleles of the Guanine nucleotide-binding protein subunit gamma-T2 (GNGT2) gene with a mCherry reporter cassette. Three-dimensional optic vesicle-like structures were produced to verify reporter fidelity and track cones throughout their development in culture. The GNGT2-T2A-mCherry hESC line faithfully and robustly labels GNGT2-expressing cones throughout the entirety of their differentiation in vitro, recapitulating normal fetal expression of this gene. Our observations indicate that human cones undergo significant migratory activity during the course of differentiation in vitro. Consistent with this, our analysis of human fetal retinae from different stages of development finds positional differences of the cone population depending on their state of maturation. This novel reporter line will provide a useful tool for investigating human cone development and disease.
Text
sxab015
- Accepted Manuscript
More information
Accepted/In Press date: 23 October 2021
e-pub ahead of print date: 19 January 2022
Published date: 26 February 2022
Additional Information:
Funding Information:
This study was supported by The Academy of Medical Sciences (Springboard Award SBF004\1038), the Gift of Sight Appeal Elkington Fellowship in Vision Sciences, Fight for Sight UK; Great Ormond Street Hospital Charity. The views expressed are those of the author(s) and not necessarily those of the funders.
Publisher Copyright:
© The Author(s) 2022. Published by Oxford University Press. All rights reserved.
Keywords:
cone photoreceptor, GNGT2, optic vesicles, retinal organoids, stem cells
Identifiers
Local EPrints ID: 456789
URI: http://eprints.soton.ac.uk/id/eprint/456789
ISSN: 1066-5099
PURE UUID: 4de298af-5a70-46ac-b943-c33e4df75088
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Date deposited: 11 May 2022 16:46
Last modified: 17 Mar 2024 07:14
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Contributors
Author:
Liliya Nazlamova
Author:
Emma Jane Cassidy
Author:
Jane C. Sowden
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