期刊
STEM CELLS
卷 39, 期 4, 页码 414-428出版社
OXFORD UNIV PRESS
DOI: 10.1002/stem.3325
关键词
cone photoreceptor; NRL; optic vesicles; retinal organoids; stem cells
资金
- Fight for Sight UK
- Great Ormond Street Hospital Charity
- Medical Research Council [MR/M015688/1]
- National Institute for Health Research
- NIHR Great Ormond Street Hospital Biomedical Research Centre
- Saudi Arabia Cultural Bureau in London
- Wellcome Trust [109053/Z/15/Z]
- Wellcome Trust [109053/Z/15/Z] Funding Source: Wellcome Trust
- MRC [MR/M015688/1] Funding Source: UKRI
The study utilized CRISPR/Cas9 gene editing to create NRL-deficient embryonic stem cell (ESC) line, which differentiated into retinal organoids. The research found that in NRL-deficient organoids, photoreceptor development exhibited abnormalities, indicating a conserved molecular switch mechanism.
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.
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