OPTIMIZING SOMATIC REPROGRAMMING FOR GENERATION OF NAÏVE INDUCED PLURIPOTENT STEM CELLS

Pluripotency is the property of an unspecialized cell to become any cell type, which is an essential feature of epiblast cells during embryogenesis. In vitro cultures of naive stem cells proved to possess the pluripotency phenotype of epiblast cells of the preimplantation stage, and they demonstrated to be powerful tools for studying early embryogenesis, epigenetics, cell lineage decisions, and extraembryonic differentiation. However, the isolation and maintenance of this cell type in culture is still an active field of research as there are no common “gold standard” protocols yet. One of the approaches to obtain naive pluripotency in vitro is through reprogramming somatic cells into human induced pluripotent stem cells (hiPSCs). Generation of naive hiPSCs from somatic cells represents a fast and efficient method but it still has some limitations, like low efficiency. Generation of low amounts of naive hiPSCs leads to the need for prolonged expansion in culture, which can result in a higher risk of chromosomal abnormalities. In this work, we optimized conditions for obtaining naive hiPSCs during the reprogramming. We customized the reprogramming medium according to the main known signaling pathways regulating naive pluripotency and, for the first time, we used feeder layers to promote the acquisition of naive pluripotency in microfluidics. We tested if there was a positive effect of feeder layers such as mouse embryonic fibroblasts or trophoblast stem cells compared to feeder-free conditions. In all conditions, naive hiPSCs were generated but the use of MEFs, rather than trophoblasts, enhanced the induction of naive hiPSCs.