Poking Pluripotency: Nanoinjection Into Human iPSCs

Abstract

Human induced pluripotent stem cells (hiPSCs) play an essential role in regenerative medicine and disease modeling, yet their utility critically depends on the efficient and safe delivery of exogenous genetic material. Conventional viral and electroporation-based methods continue to present limitations, notably biosafety and regulatory barriers, suboptimal transfection precision, and substantial post-transfection cytotoxicity. Here, for the first time, nanotube (NT)-mediated nanoinjection is introduced as an effective non-viral strategy for functional messenger RNA (mRNA) delivery into hiPSCs. To enable nanoinjection into hiPSCs, the work implements delayed extracellular matrix (ECM) application and advanced surface engineering, combined with a re-engineered NT design featuring large cargo reservoirs to increase loading capacity and sharp rim geometries for improved cell interfacing. Effective nanoinjection into hiPSCs, while maintaining their pluripotent profile, is demonstrated using mCherry, GFP, and YPet mRNA (average transfection yields: ∼55% to ∼64%), including co-transfection of mCherry and GFP mRNA (∼61%). Post-nanoinjection hiPSC integrity is confirmed over several passages by excellent pluripotency marker expression (NANOG, OCT4, SOX2) and neuronal differentiation capacity. This proof-of-concept study demonstrates that nanoinjection is a powerful tool for delivering mRNA to hiPSCs with high expression yields and establishes nanoinjection as an enabling platform for precise cellular engineering and functional stem-cell applications.