Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation.
Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation.
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DOI：
10.3389/fcell.2022.870441
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发表时间：
2022
影响因子：
5.5
通讯作者：
中科院分区：
文献类型：
--
作者：
干细胞疗法被认为是治疗视网膜功能障碍或光感受器死亡的很有前途的疗法。然而，到目前为止，适合移植的人光感受器的富集化一直是非常具有挑战性的。本研究旨在利用CRISPR/Cas9基因组编辑技术构建光感受器特异性报告基因人诱导多能干细胞(HiPSC)系，该细胞系在PAN光感受器标记Recovivin(RCVRN)的内源位点含有增强型绿色荧光蛋白(EGFP)序列。在证实成功打靶和基因稳定后，从该报告系诱导出三维视网膜器质。RCVRN-EGFP报告忠实地复制了Recoverin的内源性蛋白表达，揭示了视网膜分化过程中光感受器的发育特征。RCVRN-EGFP从光感受器前体细胞到成熟的视杆细胞和视锥细胞，都被特异性和稳定地标记。此外，荧光激活细胞分选丰富了丰富的EGFP阳性光感受器，并通过RNA测序和数据分析揭示了它们的转录组特征。此外，还提取了潜在的分化簇(CD)生物标志物，用于丰富临床应用的光感受器，如CD133用于光感受器的正向选择。综上所述，成功建立了RCVRN-EGFP报告基因HiPSC系，并构建了第一个Recoverin阳性光感受器的全球表达数据库。这些成果将为动态监测光感受器细胞发育和人光感受器的纯化提供有力的工具，从而为晚期视网膜疾病的光感受器细胞治疗提供便利。
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.
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影响因子：
5.9
作者：
Gagliardi G;Ben M'Barek K;Chaffiol A;Slembrouck-Brec A;Conart JB;Nanteau C;Rabesandratana O;Sahel JA;Duebel J;Orieux G;Reichman S;Goureau O
通讯作者：
Goureau O
DOI：
10.1002/stem.2051
发表时间：
2015-08
期刊：
Stem cells (Dayton, Ohio)
影响因子：
--
作者：
Lakowski J;Gonzalez-Cordero A;West EL;Han YT;Welby E;Naeem A;Blackford SJ;Bainbridge JW;Pearson RA;Ali RR;Sowden JC
通讯作者：
Sowden JC
DOI：
10.1002/stem.2775
发表时间：
2018-05
期刊：
Stem cells (Dayton, Ohio)
影响因子：
--
作者：
Lakowski J;Welby E;Budinger D;Di Marco F;Di Foggia V;Bainbridge JWB;Wallace K;Gamm DM;Ali RR;Sowden JC
通讯作者：
Sowden JC
影响因子：
1.3
作者：
Haybar, Habib;Behzad, Masumeh Maleki;Saki, Najmaldin
通讯作者：
Saki, Najmaldin
影响因子：
7.5
作者：
Lamb MG;Rangarajan HG;Tullius BP;Lee DA
通讯作者：
Lee DA