GNAT2-EGFP视网膜类器官中视锥光感受器成熟的间歇性实时成像。

Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids.

作者信息

Bai Jinlun, Koos David S, Stepanian Kayla, Fouladian Zachary, Shayler Dominic W H, Aparicio Jennifer G, Fraser Scott E, Moats Rex A, Cobrinik David

机构信息

The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, CA, USA.

The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, CA, USA.

出版信息

bioRxiv. 2023 Mar 1:2023.02.28.530518. doi: 10.1101/2023.02.28.530518.

DOI:10.1101/2023.02.28.530518

PMID:36909527

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10002746/

Abstract

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 (). In retinal organoids generated from these iPSCs, the GNAT2-EGFP allele robustly and exclusively labeled both immature and mature cones starting at culture day 34. 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.

摘要

荧光报告多能干细胞（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有力工具。当与活体成像相结合时，它们能够直接且反复地观察发育中的视网膜组织内的细胞行为。在此，我们通过在WTC11-mTagRFPT-LMNB1人诱导多能干细胞（iPSC）中进行CRISPR/Cas9基因组编辑，在（）的N端插入增强型绿色荧光蛋白（EGFP）编码序列和一个2A自切割肽，从而构建了一个人类视锥光感受器报告细胞系。在由这些iPSC产生的视网膜类器官中，GNAT2-EGFP等位基因从培养第34天开始就强烈且特异性地标记未成熟和成熟的视锥细胞。对水凝胶固定的视网膜类器官进行的间歇性共聚焦活体成像能够追踪单个视锥细胞超过18周的形态成熟过程，并揭示了线粒体在内段的积累以及从第126天到第153天每天以12.2立方微米的速度生长。固定的GNAT2-EGFP视锥细胞报告类器官为研究人类视锥细胞发育和疾病提供了一个有价值的工具。