Generation of Induced Pluripotent Stem Cells from Somatic Cells via Solid Lipid Nanoparticle-Mediated Plasmid DNA Delivery.

Abstract

Induced pluripotent stem cells (iPSCs) have enabled significant advances in regenerative medicine; however, reprogramming methods using viral vectors pose risks to safety and genomic integrity in clinical applications. This protocol details a non-viral, integration-free approach for delivering plasmid DNA (pDNA) into somatic cells via solid lipid nanoparticles (SLNs). SLNs protect pDNA from enzymatic degradation and ensure high transfection efficiency in cells, thereby preventing permanent genetic alterations in the host genome.The protocol provides step-by-step guidance for the preparation of human fibroblasts, the sequential transcription cycles of SLN-pDNA encoding OSKM factors, and the induction and isolation of iPSC colonies. Furthermore, comprehensive characterization methods are described to confirm pluripotency, including monitoring GFP expression levels, quantitative real-time PCR, immunofluorescence staining, flow cytometry, alkaline phosphatase activity assays, and in vitro tri-lineage differentiation.iPSCs obtained while preserving genomic integrity can be safely used in advanced regenerative medicine applications, such as disease modeling, high-throughput drug screening, and personalized cell therapies. This approach enhances safety and efficacy in clinical settings and provides a valuable platform for the development of iPSC-based therapeutic and research applications.