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Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation
Stem cell-based cell therapies are considered to be promising treatments for retinal disorders with dysfunction or death of photoreceptors. However, the enrichment of human photoreceptors suitable for transplantation has been highly challenging so far. This study aimed to generate a photoreceptor-sp...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9096726/ https://www.ncbi.nlm.nih.gov/pubmed/35573687 http://dx.doi.org/10.3389/fcell.2022.870441 |
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author | Guan, Yuanyuan Wang, Yuan Zheng, Dandan Xie, Bingbing Xu, Ping Gao, Guanjie Zhong, Xiufeng |
author_facet | Guan, Yuanyuan Wang, Yuan Zheng, Dandan Xie, Bingbing Xu, Ping Gao, Guanjie Zhong, Xiufeng |
author_sort | Guan, Yuanyuan |
collection | PubMed |
description | Stem cell-based cell therapies are considered to be promising treatments for retinal disorders with dysfunction or death of photoreceptors. However, the enrichment of human photoreceptors suitable for transplantation has been highly challenging so far. This study aimed to generate a photoreceptor-specific reporter human induced pluripotent stem cell (hiPSC) line using CRISPR/Cas9 genome editing, which harbored an enhanced green fluorescent protein (eGFP) sequence at the endogenous locus of the pan photoreceptor marker recoverin (RCVRN). After confirmation of successful targeting and gene stability, three-dimensional retinal organoids were induced from this reporter line. The RCVRN-eGFP reporter faithfully replicated endogenous protein expression of recoverin and revealed the developmental characteristics of photoreceptors during retinal differentiation. The RCVRN-eGFP specifically and steadily labeled photoreceptor cells from photoreceptor precursors to mature rods and cones. Additionally, abundant eGFP-positive photoreceptors were enriched by fluorescence-activated cell sorting, and their transcriptome signatures were revealed by RNA sequencing and data analysis. Moreover, potential clusters of differentiation (CD) biomarkers were extracted for the enrichment of photoreceptors for clinical applications, such as CD133 for the positive selection of photoreceptors. Altogether, the RCVRN-eGFP reporter hiPSC line was successfully established and the first global expression database of recoverin-positive photoreceptors was constructed. These achievements will provide a powerful tool for dynamically monitoring photoreceptor cell development and purification of human photoreceptors, thus facilitating photoreceptor cell therapy for advanced retinal disorders. |
format | Online Article Text |
id | pubmed-9096726 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-90967262022-05-13 Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation Guan, Yuanyuan Wang, Yuan Zheng, Dandan Xie, Bingbing Xu, Ping Gao, Guanjie Zhong, Xiufeng Front Cell Dev Biol Cell and Developmental Biology Stem cell-based cell therapies are considered to be promising treatments for retinal disorders with dysfunction or death of photoreceptors. However, the enrichment of human photoreceptors suitable for transplantation has been highly challenging so far. This study aimed to generate a photoreceptor-specific reporter human induced pluripotent stem cell (hiPSC) line using CRISPR/Cas9 genome editing, which harbored an enhanced green fluorescent protein (eGFP) sequence at the endogenous locus of the pan photoreceptor marker recoverin (RCVRN). After confirmation of successful targeting and gene stability, three-dimensional retinal organoids were induced from this reporter line. The RCVRN-eGFP reporter faithfully replicated endogenous protein expression of recoverin and revealed the developmental characteristics of photoreceptors during retinal differentiation. The RCVRN-eGFP specifically and steadily labeled photoreceptor cells from photoreceptor precursors to mature rods and cones. Additionally, abundant eGFP-positive photoreceptors were enriched by fluorescence-activated cell sorting, and their transcriptome signatures were revealed by RNA sequencing and data analysis. Moreover, potential clusters of differentiation (CD) biomarkers were extracted for the enrichment of photoreceptors for clinical applications, such as CD133 for the positive selection of photoreceptors. Altogether, the RCVRN-eGFP reporter hiPSC line was successfully established and the first global expression database of recoverin-positive photoreceptors was constructed. These achievements will provide a powerful tool for dynamically monitoring photoreceptor cell development and purification of human photoreceptors, thus facilitating photoreceptor cell therapy for advanced retinal disorders. Frontiers Media S.A. 2022-04-28 /pmc/articles/PMC9096726/ /pubmed/35573687 http://dx.doi.org/10.3389/fcell.2022.870441 Text en Copyright © 2022 Guan, Wang, Zheng, Xie, Xu, Gao and Zhong. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Cell and Developmental Biology Guan, Yuanyuan Wang, Yuan Zheng, Dandan Xie, Bingbing Xu, Ping Gao, Guanjie Zhong, Xiufeng Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation |
title | Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation |
title_full | Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation |
title_fullStr | Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation |
title_full_unstemmed | Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation |
title_short | Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation |
title_sort | generation of an rcvrn-egfp reporter hipsc line by crispr/cas9 to monitor photoreceptor cell development and facilitate the cell enrichment for transplantation |
topic | Cell and Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9096726/ https://www.ncbi.nlm.nih.gov/pubmed/35573687 http://dx.doi.org/10.3389/fcell.2022.870441 |
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