Senior Director Thermo Fisher Scientific Carlsbad, California, United States
Abstract: Induced pluripotent stem cell (iPSC) therapies hold significant promise for regenerative medicine, particularly for treating degenerative diseases and serving as a renewable source for allogeneic cell therapies. Efficient and footprint-free iPSC generation systems, like RNA-based reprogramming, are preferential due to their transient nature and eliminating the need for residual factor clearance. However, broader application of RNA reprogramming has been impeded by difficulties in efficiently delivering RNA to starting materials like blood cells. To that aim, the Solupore platform offers a solution using physicochemical processes to temporarily permeabilize cell membranes, ensuring effective cargo delivery while maintaining cell health for non-viral delivery. This non-invasive approach has shown superior cell viability when compared to traditional electroporation, and successful delivery to various cell types. In this study, the Solupore was utilized to reprogram CD34+, T cells, and PBMCs with both messenger RNA (mRNA) and self-replicating RNA (srRNA). Repeated transfections resulted in high cell viability and recovery, and successful iPSC generation. iPSC clones exhibiting typical morphology were cultured for at least five passages before undergoing characterization. Pluripotency was verified using flow cytometry and immunocytochemistry. Embryoid bodies were formed to assess tri-lineage differentiation potential. Select clones were then further modified using the Solupore for CRISPR-Cas9 delivery. This pioneering study demonstrates the successful generation and modification of iPSCs from blood cells using RNA delivery, thereby broadening the range of starting cells, reprogramming techniques, and delivery methods, and underscoring its potential in cell therapy applications.
