Transduction of AAV Vectors in iPSC-Derived Retinal Organoids

Stem cell-derived organoids research provides an exciting model and technology to develop new therapies for difficult and intractable diseases. Human-induced pluripotent stem cells (hiPSCs) have the potential to differentiate into organ-like three-dimensional structures, especially when it is challenging to obtain patient samples for this purpose. This chapter outlines a comprehensive differentiation protocol for the generation and manipulation of retinal organoids derived from pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and hiPSCs. The method involves culturing PSCs, differentiating them into retinal organoids, and in vitro transduction of organoids with adeno-associated virus (AAV) vectors. AAVs can be used to label a desired population, to test novel promoters, and to test gene therapies. We will also describe key steps following organoid transduction, which entail collection, immunostaining, imaging and preservation for molecular biology readouts. The protocol provides a detailed guide for researchers working in the field of retinal biology and gene therapy.