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
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DOI：
10.1093/stmcls/sxab015
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发表时间：
2022-02-26
期刊：
影响因子：
5.2
通讯作者：
Lakowski, Jorn
中科院分区：
文献类型：
--
作者：
Nazlamova, Liliya;Cassidy, Emma-Jane;Lakowski, Jorn
人类多能干细胞中产生的荧光报告基因系是追踪、分离和分析活体培养物中的细胞类型和谱系的非常有用的工具。在这里，我们通过对人类胚胎干细胞 (hESC) 系进行 CRISPR/Cas9 基因组编辑，生成第一个人类视锥光感受器报告细胞系，用 mCherry 报告盒标记鸟嘌呤核苷酸结合蛋白亚基 gamma-T2 (GNGT2) 基因的两个等位基因。生产了三维视囊泡样结构，以验证报告基因的保真度并在培养过程中跟踪视锥细胞的整个发育过程。 GNGT2-T2A-mCherry hESC 系在表达 GNGT2 的视锥细胞的整个体外分化过程中忠实而可靠地标记，重现了该基因的正常胎儿表达。我们的观察表明，人类视锥细胞在体外分化过程中经历了显着的迁移活动。与此一致的是，我们对不同发育阶段的人类胎儿视网膜的分析发现视锥细胞群的位置差异取决于其成熟状态。这种新颖的报告线将为研究人类视锥细胞的发育和疾病提供有用的工具。
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.