Project description:Emergence of induced pluripotent stem cells (iPSC) technology has paved novel routes for regenerative medicine. iPSCs offer the possibilities of disease modeling, drug toxicity studies as well as cell replacement therapies by autologous transplantation. Classical protocols of iPSC generation harness infection by retro- or lenti-viruses. Although such integrating viruses represent very robust tools for reprogramming, the presence of viral transgenes in iPSCs is deleterious as it holds the risk of insertional mutagenesis leading to malignant transformation. Moreover, remaining reprogramming transgenes have been shown to affect the differentiation potential of iPSCs. More recently, alternative protocols have been explored to derive transgene-free iPSC, including use of transposons, mRNA transfection, episomal plasmid transfection, and infection with non-integrating viruses such as Sendai virus. However, the utility of such protocols remains limited due to low efficiency and narrow range of cell specificity. In this study we aim at combining the robustness of lentiviral reprogramming with the high efficacy of Cre recombinase protein transduction to readily delete reprogramming transgenes from iPSCs. We demonstrate rapid generation of transgene-free human iPSCs by excising the loxP-flanked reprogramming cassette employing direct delivery of biologically active Cre protein. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs do resemble more to human ESCs and has better differentiation potential than iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of superior transgene-free iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies. mRNA extracted from human Fibroblasts (AR1034ZIMA), human Embryonic Stem Cell line I3 (hES I3), three human induced Pluripotent Stem Cell clones 1, 1.2 and 1.4 (fl-ARiPS cl1, del-ARiPS cl 1.2, del-ARiPS cl1.4) has been hybridized on Illumina Human HT-12 (version 4 revision 2) arrays for genome wide expression analysis. Samples were run at least as duplicate technical replicates. Differential gene expression analysis has been performed on the grouped expression data with the human embryonic stem cells (hES I3) group as the reference.

Project description:Human induced pluripotent stem cells (hiPSCs) hold substantial promise for regenerative medicine and cell manufacturing, yet their widespread adoption remains limited by reprogramming strategies that are inefficient, costly, unsafe, and difficult to scale, driven by reliance on viral vectors, limited virus-free gene delivery technologies, and two-dimensional (2D) culture systems lacking microenvironmental control. Here, we present a virus-free, materials-guided reprogramming platform that integrates engineered polymeric nanoparticles with a biomimetic three-dimensional (3D) scaffold to jointly regulate gene delivery and cell-state transitions during fate conversion. A rationally designed fluorocarbon- and heparin-modified polymer nanoparticle enables efficient, low-toxicity episomal delivery of reprogramming factors, while a porous chitosan scaffold provides a permissive 3D microenvironment that accelerates reprogramming kinetics and suppresses somatic cell overgrowth. This integrated strategy concurrently improves reprogramming efficiency, kinetics, and workflow simplicity without the use of viral vectors or manual colony isolation. Scaffold-supported reprogramming yields an approximately eightfold increase in SSEA-4⁺/TRA-1-60⁺ cells by day 25 compared with non-viral 2D conditions, with pluripotent populations emerging as early as day 7, enabling hiPSC generation within weeks rather than the months typically required by conventional workflows. Transcriptomic analyses reveal that the 3D scaffold functions as an active regulatory element, suppressing inflammatory and extracellular matrix–associated programs that stabilize somatic identity while promoting chromatin-remodeling and stem-cell-associated pathways. Importantly, the platform supports a continuous, selection-free workflow, enabling direct differentiation of hiPSCs within the scaffold and further reducing processing time. By coupling delivery chemistry with microenvironmental control, this nanoparticle–scaffold platform provides a safe, efficient, and scalable route for hiPSC generation with direct relevance to regenerative medicine and patient-specific cell manufacturing.

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