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 cellular processes. Conventional genetic engineering of hiPSCs requires laborious processes involving transfection, selection, and expansion that can cause karyotypic abnormalities or transgene silencing during differentiation. The use of self-amplifying RNA (saRNA) 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 demonstrated that protein expression can persist for weeks. Specifically, saRNA delivery enabled highly efficient forward programming to Ngn2-induced neurons and measurement of functional reporters over time. We show that a single transfection of saRNA-encoded jRCaMP1b in hiPSCs generates sustained reporter expression throughout differentiation to 3D cardiac spheroids, allowing the tracking of cardiomyocyte function and drug responses. Altogether, our systematic analysis shows that saRNA extends transgene expression in hiPSCs, supporting integration-free cell fate programming and functional reporter measurement in clinically relevant model systems.