Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids.
Jinlun BaiDavid S KoosKayla StepanianZachary FouladianDominic W H ShaylerJennifer G AparicioScott E FraserRex A MoatsDavid CobrinikPublished in: Disease models & mechanisms (2023)
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.
Keyphrases
- induced pluripotent stem cells
- crispr cas
- genome editing
- optical coherence tomography
- diabetic retinopathy
- high resolution
- stem cells
- optic nerve
- binding protein
- single cell
- computed tomography
- drug delivery
- cell therapy
- ionic liquid
- living cells
- bone marrow
- hyaluronic acid
- single molecule
- mesenchymal stem cells
- capillary electrophoresis
- wound healing
- tissue engineering