Poking Pluripotency: Nanoinjection Into Human iPSCs

Area of Science:

• Biotechnology and Regenerative Medicine
• Cellular Engineering
• Nanotechnology

Background:

• Human induced pluripotent stem cells (hiPSCs) are crucial for regenerative medicine and disease modeling.
• Current genetic material delivery methods (viral, electroporation) have safety, precision, and cytotoxicity limitations.
• Efficient and safe delivery of exogenous genetic material into hiPSCs is essential for their application.

Purpose of the Study:

• To introduce nanotube (NT)-mediated nanoinjection as a novel, non-viral strategy for functional messenger RNA (mRNA) delivery into hiPSCs.
• To demonstrate the efficacy and safety of NT-mediated nanoinjection in hiPSCs, preserving their pluripotent state.

Main Methods:

• Developed re-engineered NTs with enhanced cargo capacity and cell interfacing geometry.
• Implemented delayed extracellular matrix (ECM) application and advanced surface engineering for hiPSC nanoinjection.
• Delivered various mRNA payloads (mCherry, GFP, YPet) into hiPSCs via nanoinjection.

Main Results:

• Achieved effective nanoinjection into hiPSCs with average transfection yields of ~55% to ~64% for single mRNAs.
• Demonstrated successful co-transfection of mCherry and GFP mRNA with yields of ~61%.
• Confirmed hiPSC integrity and pluripotency post-nanoinjection through marker expression (NANOG, OCT4, SOX2) and neuronal differentiation capacity over several passages.

Conclusions:

• NT-mediated nanoinjection is a powerful and effective non-viral tool for delivering mRNA into hiPSCs.
• This method maintains hiPSC pluripotency and functional differentiation capabilities.
• Nanoinjection serves as an enabling platform for precise cellular engineering and advanced stem cell applications.