Pluristyx, Inc. Seattle, Washington, United States
Abstract: Induced Pluripotent Stem Cells (iPSCs) are a transformative and ethical alternative to Embryonic Stem Cells for regenerative medicine, disease modeling, and drug discovery. Their ability to be derived from somatic cells and differentiated into various lineages makes them a versatile platform for developing stem-cell-based therapies. Most reprogramming methods are low efficiency and require early clonal isolation to generate iPSC, resulting in high passage number and limited genetic diversity that negatively impact efficiency, safety and robustness. These limitations increase production costs and timelines, hindering the scalability of iPSCs for clinical use. We developed a novel, efficient, footprint-free mRNA-based reprogramming workflow to address these gaps. Here we demonstrate that this technology safely reprograms fibroblasts and mesenchymal stromal cells with efficiencies as high as 20%, enabling bulk, polyclonal reprogramming and reducing reliance or eliminating the need for early clone selection. Clone selection is deferred to gene editing or Master Cell Bank (MCB) stages, reducing culture bottlenecks and genome stress, minimizing population doublings and passage numbers, while preserving genetic and epigenetic diversity. Optimization of this workflow across six compliant cell lines highlights its robustness and scalability. Results demonstrate a stable polyclonal population suitable for downstream gene editing and processing, reducing timelines and manufacturing costs. Enhanced in-process and release testing ensure manufacturing of iPSCs with safety and quality for both allogeneic and autologous applications. This platform represents a major advancement in iPSC manufacturing, overcoming critical barriers in cost, safety, and efficiency. Its broad applicability in cell therapy, disease modeling, and drug screening positions it as a pivotal technology for accelerating iPSC-based research and autologous and allogeneic clinical therapies.
