Nanopore-Based Electroporation Enables High-Efficiency, Rapid RNA-Mediated Reprogramming of Primary Fibroblasts into Human iPSCs

Induced pluripotent stem cells (iPSCs) have revolutionized regenerative medicine by overcoming the ethical and immunological challenges posed by embryonic stem cells, while nonviral reprogramming of fibroblasts to iPSCs often faces challenges in efficiency and complexity. We present a nanopore electroporation (NanoEP) system for efficient delivery of circular RNA (circRNA) encoding six reprogramming factors (OCT4, SOX2, KLF4, c-MYC, LIN28A, and NANOG) into human fibroblasts. NanoEP, employing engineered polycarbonate membranes for localized permeabilization, achieves >90% transfection efficiency at low voltages, enhancing cell viability and minimizing proinflammatory cytokine release (IL-6 and IL-18) compared to other methods. Repeated NanoEP-mediated circRNA delivery yields >100% reprogramming efficiency and the rapid establishment of stable iPSC lines within 10 days. These results highlight NanoEP as a rapid, efficient, and safe nonviral circRNA reprogramming strategy, enabling advancements in disease modeling, drug development, and cell-based therapies.