荧光报告基因多能干细胞 (PSC) 衍生的视网膜类器官是研究细胞类型特异性发育和疾病表型的强大工具。当与实时成像相结合时，它们能够直接、重复地观察发育中的视网膜组织内的细胞行为。在这里，我们通过在 WTC11-mTagRFPT-LMNB1 人类诱导多能干细胞 (iPSC) 的 CRISPR/Cas9 基因组编辑中插入增强型绿色荧光蛋白 (EGFP) 编码序列和 2A 自切割肽，生成了人类视锥光感受器报告系。鸟嘌呤核苷酸结合蛋白亚基 Alpha 转导蛋白 2 (GNAT2) 的 N 末端。在这些 iPSC 生成的视网膜类器官中，GNAT2-EGFP 等位基因强烈且专门地标记未成熟和成熟的视锥细胞。水凝胶固定化视网膜类器官的情景共聚焦实时成像可以在超过 18 周的时间内追踪单个视锥细胞的形态成熟，并揭示线粒体内节的积累以及从第 126 天到第 153 天每天以 12.2 立方微米的速度生长。固定化 GNAT2-EGFP 视锥细胞报告类器官为研究人体锥体发育和疾病提供了宝贵的工具。© 2023。由 The Company of Biologies Ltd 出版。

Fluorescent reporter pluripotent stem cell (PSC) derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus of Guanine Nucleotide-Binding Protein Subunit Alpha Transducin 2 (GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP alleles robustly and exclusively labeled both immature and mature cones. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of morphological maturation of individual cones for >18 weeks and revealed inner segment accumulation of mitochondria and growth at 12.2 cubic microns per day from day 126 to day 153. Immobilized GNAT2-EGFP cone reporter organoids provide a valuable tool for investigating human cone development and disease.© 2023. Published by The Company of Biologists Ltd.