Self-amplifying RNA enables rapid, durable, integration-free programming of hiPSCs

Genetic modification of human induced pluripotent stem cells (hiPSCs) is a powerful approach to measure and manipulate the cellular processes underlying differentiation and disease. Conventional genetic engineering of hiPSC lines requires a laborious process involving transfection, selection and expansion that can result in karyotypic abnormalities or transgene silencing during differentiation, limiting their applications. Self-amplifying RNA (saRNA) delivery is a potential alternative integration-free method for durable expression of transgenes. Here, we used saRNA to deliver transcription factors and functional reporters in hiPSCs and demonstrate that expression can persist for weeks. Specifically, saRNA delivery enables highly efficient forward programming to Ngn2-induced neurons and enables measurement of functional reporters over time. We show that a single transfection of saRNA encoded jRCaMP1b reporter in hiPSCs generates sustained expression throughout differentiation to 3D cardiac spheroids. The persistence of the reporter allows measurement of calcium dynamics at a single-cell and population level over weeks, allowing tracking of cardiomyocyte maturation and drug responses. Together, our systematic analysis shows that saRNA provides sustained transgene expression in hiPSCs, supporting integration- free cell-fate programming and measurement of functional reporters in clinically relevant model systems.