NRL−/− gene edited human embryonic stem cells generate rod‐deficient retinal organoids enriched in S‐cone‐like photoreceptors
NRL−/− gene edited human embryonic stem cells generate rod‐deficient retinal organoids enriched in S‐cone‐like photoreceptors
Organoid cultures represent a unique tool to investigate the developmental complexity of tissues like the human retina. NRL is a transcription factor required for the specification and homeostasis of mammalian rod photoreceptors. In Nrl-deficient mice, photoreceptor precursor cells do not differentiate into rods, and instead follow a default photoreceptor specification pathway to generate S-cone-like cells. To investigate whether this genetic switch mechanism is conserved in humans, we used CRISPR/Cas9 gene editing to engineer an NRL-deficient embryonic stem cell (ESC) line (NRL
−/−), and differentiated it into retinal organoids. Retinal organoids self-organize and resemble embryonic optic vesicles (OVs) that recapitulate the natural histogenesis of rods and cone photoreceptors. NRL
−/− OVs develop comparably to controls, and exhibit a laminated, organized retinal structure with markers of photoreceptor synaptogenesis. Using immunohistochemistry and quantitative polymerase chain reaction (qPCR), we observed that NRL
−/− OVs do not express NRL, or other rod photoreceptor markers directly or indirectly regulated by NRL. On the contrary, they show an abnormal number of photoreceptors positive for S-OPSIN, which define a primordial subtype of cone, and overexpress other cone genes indicating a conserved molecular switch in mammals. This study represents the first evidence in a human in vitro ESC-derived organoid system that NRL is required to define rod identity, and that in its absence S-cone-like cells develop as the default photoreceptor cell type. It shows how gene edited retinal organoids provide a useful system to investigate human photoreceptor specification, relevant for efforts to generate cells for transplantation in retinal degenerative diseases.
NRL, cone photoreceptor, optic vesicles, retinal organoids, stem cells
414-428
Cuevas, Elisa
896f2004-610c-4e77-bed7-cbcd00bf49a6
Holder, Daniel L.
c4c062be-7744-4d1d-9f04-15664830a23f
Alshehri, Ashwak H.
b26f8729-af05-4d8f-b1d9-7688948613bc
Tréguier, Julie
beabe1e0-8a42-419b-82eb-8fff8b3c5307
Lakowski, Jorn
1856e739-982a-412a-87c7-abf1610f5384
Sowden, Jane C.
6f414289-9a33-42ad-a3cd-2fa828b7ac27
April 2021
Cuevas, Elisa
896f2004-610c-4e77-bed7-cbcd00bf49a6
Holder, Daniel L.
c4c062be-7744-4d1d-9f04-15664830a23f
Alshehri, Ashwak H.
b26f8729-af05-4d8f-b1d9-7688948613bc
Tréguier, Julie
beabe1e0-8a42-419b-82eb-8fff8b3c5307
Lakowski, Jorn
1856e739-982a-412a-87c7-abf1610f5384
Sowden, Jane C.
6f414289-9a33-42ad-a3cd-2fa828b7ac27
Cuevas, Elisa, Holder, Daniel L., Alshehri, Ashwak H., Tréguier, Julie, Lakowski, Jorn and Sowden, Jane C.
(2021)
NRL−/− gene edited human embryonic stem cells generate rod‐deficient retinal organoids enriched in S‐cone‐like photoreceptors.
Stem Cells, 39 (4), .
(doi:10.1002/stem.3325).
Abstract
Organoid cultures represent a unique tool to investigate the developmental complexity of tissues like the human retina. NRL is a transcription factor required for the specification and homeostasis of mammalian rod photoreceptors. In Nrl-deficient mice, photoreceptor precursor cells do not differentiate into rods, and instead follow a default photoreceptor specification pathway to generate S-cone-like cells. To investigate whether this genetic switch mechanism is conserved in humans, we used CRISPR/Cas9 gene editing to engineer an NRL-deficient embryonic stem cell (ESC) line (NRL
−/−), and differentiated it into retinal organoids. Retinal organoids self-organize and resemble embryonic optic vesicles (OVs) that recapitulate the natural histogenesis of rods and cone photoreceptors. NRL
−/− OVs develop comparably to controls, and exhibit a laminated, organized retinal structure with markers of photoreceptor synaptogenesis. Using immunohistochemistry and quantitative polymerase chain reaction (qPCR), we observed that NRL
−/− OVs do not express NRL, or other rod photoreceptor markers directly or indirectly regulated by NRL. On the contrary, they show an abnormal number of photoreceptors positive for S-OPSIN, which define a primordial subtype of cone, and overexpress other cone genes indicating a conserved molecular switch in mammals. This study represents the first evidence in a human in vitro ESC-derived organoid system that NRL is required to define rod identity, and that in its absence S-cone-like cells develop as the default photoreceptor cell type. It shows how gene edited retinal organoids provide a useful system to investigate human photoreceptor specification, relevant for efforts to generate cells for transplantation in retinal degenerative diseases.
Text
Elisa Cuevas
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More information
Accepted/In Press date: 24 November 2020
e-pub ahead of print date: 5 January 2021
Published date: April 2021
Additional Information:
©2021 The Authors. Stem Cells published by Wiley Periodicals LLC on behalf of AlphaMed Press 2021.
Keywords:
NRL, cone photoreceptor, optic vesicles, retinal organoids, stem cells
Identifiers
Local EPrints ID: 447395
URI: http://eprints.soton.ac.uk/id/eprint/447395
ISSN: 1066-5099
PURE UUID: 5fedf72f-3dff-40b7-aac7-6a628ded8a23
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Date deposited: 10 Mar 2021 17:43
Last modified: 17 Mar 2024 03:48
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Contributors
Author:
Elisa Cuevas
Author:
Daniel L. Holder
Author:
Ashwak H. Alshehri
Author:
Julie Tréguier
Author:
Jane C. Sowden
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