Generation and Characterization of Patient-Derived Human Pluripotent Stem Cell Lines for Retinal Gene Therapy

Patient-derived human-induced pluripotent stem cells (hiPSCs) are a valuable tool for validation of retinal gene therapies. These cells exhibit features identical to human embryonic stem cells (hESCs), such as self-renewal and pluripotency, and can differentiate into various retinal cell types, making them ideal for assessing clinically compliant viral vectors in a human retinal context. Several reprogramming methods have been developed, with integration-free methods being preferable due to their low risk of genomic integration and suitability for clinical applications. This chapter details the generation and characterization of patient-derived hiPSC lines from peripheral blood mononuclear cells (PBMCs) using non-integrative Sendai virus, with a focus on the thorough quality control (QC) necessary post-reprogramming. The QC includes confirming the expression of undifferentiated stem cell markers, the ability to differentiate into the three germ layers, and the absence of chromosomal abnormalities. This protocol provides a stepwise procedure for generating hiPSCs using an integration-free approach in 4 weeks.