Human induced pluripotent stem cells (hiPSCs) play a crucial role in regenerative medicine and disease modeling, but their efficacy hinges on the efficient and safe delivery of exogenous genetic material. Traditional viral and electroporation methods still have limitations, especially in terms of biosafety and regulatory barriers, poor transfection accuracy, and high cytotoxicity after transfection. Here, the introduction of nanotube (NT)-mediated nanoinjection is presented as an effective non-viral strategy for delivering functional messenger RNA (mRNA) to high pluripotent stem cells (hiPSCs). To achieve nanoinjection into hiPSCs, this study implemented delayed extracellular matrix (ECM) application and advanced surface engineering, combined with a redesigned NT design, using large cargo tanks to enhance loading capacity and sharp edge geometries to improve the cell interface. Through mCherry, GFP, and YPet mRNA (average transfection yield: approximately 55% to ∼64%), mRNA was effectively injected into high pluripotent stem cells via nanoinjection, including co-transfection of mCherry and GFP mRNA (approximately 61%). The excellent pluripotency marker expression (NANOG, OCT4, SOX2) and neuronal differentiation ability through multiple channels confirmed the integrity of high pluripotent cells after nanoinjection. This proof-of-concept study demonstrates that nanoinjection is a powerful tool for delivering mRNA to high expression yield hiPSCs and establishes nanoinjection as a platform for precise cell engineering and functional stem cell applications. This research was published under the title "Poking Pluripotency: Nanoinjection Into Human iPSCs" in Advanced Materials.
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DOI: 10.1002/adma.202521046