Generating Human Induced Pluripotent Stem Cell Via Low-Dose Polyethylenimine-Mediated Transfection: An Optimized...

Area of Science:

• Stem Cell Biology
• Gene Delivery Systems
• Cellular Reprogramming

Background:

• Generating human induced pluripotent stem cells (hiPSCs) is crucial for regenerative medicine.
• Existing reprogramming methods often involve expensive reagents and specialized equipment.
• Polyethylenimine (PEI)-mediated transfection offers a potentially more accessible alternative for hiPSC generation.

Purpose of the Study:

• To optimize PEI-mediated transfection conditions for human dermal fibroblasts (HDFs).
• To establish a cost-effective and efficient protocol for generating hiPSCs from HDFs.
• To evaluate the impact of various transfection parameters on efficiency and cytotoxicity.

Main Methods:

• Utilized PEI reagent to deliver oriP/EBNA1-based vectors into HDFs.
• Optimized parameters including N/P ratio, DNA/PEI concentrations, serum presence, and starvation duration.
• Assessed transfection efficiency and cytotoxicity using flow cytometry and fluorescent microscopy.

Main Results:

• Identified optimal conditions for PEI transfection of HDFs with low cytotoxicity.
• Achieved high transfection efficiency (∼40% GFP-positive HDFs) at an N/P ratio of 3.
• Successfully generated and characterized hiPSC lines using the optimized protocol.

Conclusions:

• 25 kDa branched PEI enables efficient transfection of HDFs for hiPSC generation.
• The developed protocol is simple, cost-effective, and requires no specialized equipment.
• This method provides a viable alternative for widespread hiPSC generation.