Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) represents a significant advance in stem cell biology, with enormous potential to deepen the understanding of the molecular mechanisms of development, as well as to expand the current possibilities of cell therapies. Unlike embryonic stem cells (ESCs), iPSCs circumvent ethical issues by eliminating the need for embryo destruction, and they allow a personalized approach, as they can be obtained directly from the individual’s own cells for disease modeling and the development of new therapies. Conventional reprogramming methods generate “primed” human iPSCs, similar to the cells present at the late blastocyst stage. These cells preserve somatic epigenetic memory and, in female cells, are in a post-X-chromosome-inactivation (XCI) state, with one active X chromosome and one inactive (XaXi). Recent advances have enabled the generation of “naïve” iPSCs, comparable to ESCs of the early blastocyst. “Naïve” iPSCs offer significant advantages, including greater pluripotency, a broader differentiation potential, less somatic epigenetic memory, and, in the case of female cells, a pre-XCI state with both X chromosomes active (XaXa). Reprogramming into “naïve” iPSCs continues to be refined to increase efficiency and generate high-quality stem cells. However, the molecular mechanisms that regulate the transition to the “naïve” state are still not completely understood. A crucial step in this process is the reversal of XCI in female cells. XCI is an epigenetic mechanism in which one of the X chromosomes is silenced by the long non-coding RNA XIST, balancing the expression of X-linked genes between XX and XY individuals. During reprogramming to the “naïve” state, this process is reversed, requiring extensive epigenetic remodeling to reactivate the inactive X chromosome (Xi). Although the mechanisms of XCI reversal have been studied during the reprogramming of mouse iPSCs, differences between species suggest that the dynamics of XCI in humans may be distinct and remain to be explored. This project investigates the reversal of XCI during reprogramming to the “naïve” state, with the aim of clarifying the molecular mechanisms involved in this transition and optimizing the generation of high-quality iPSCs. To this end, a fluorescent reporter system will be developed and tested to monitor the reversal of XCI under normal and perturbed reprogramming conditions, drawing on advanced techniques of CRISPR-Cas9 gene editing, cell and molecular biology, and multi-omics approaches. First, the active (Xa) and inactive (Xi) X chromosomes will be marked with distinct fluorescent reporters at the HUWE1 gene in “primed” female ESCs (XaXi) using CRISPR-Cas9. These cells will subsequently be differentiated into XaXi fibroblasts, which will serve as donor somatic cells for the reprogramming. Next, these reporter fibroblasts will be reprogrammed into “naïve” iPSCs. The reporter system for X chromosome activity will allow tracking of HUWE1 reactivation on the Xi and isolation of cells for RNA-seq analysis. Finally, we will perturb the system by transiently reducing XIST expression in the early phases of reprogramming, through modified antisense oligonucleotides. We will assess whether this intervention facilitates the reversal of XCI and influences the conversion to “naïve” XaXa iPSCs. This assay will allow us not only to understand the role of XCI reversal in the acquisition of “naïve” identity, but also to validate the reporter system for X chromosome activity as a tool to optimize future reprogramming protocols. In summary, this project will deepen the knowledge of XCI reversal during “naïve” reprogramming, contributing to the improvement of current protocols and to the generation of high-quality iPSCs for research and cell therapies. Thus, it directly supports Goal 3 of the 2030 Agenda for Sustainable Development: “Good Health and Well-being,” increasing the safety and applicability of iPSCs in personalized regenerative medicine. The feasibility of the project rests on the technical expertise of the team, reinforced by collaboration with expert consultants in key areas. Their support strengthens the success of this innovative work, opening a new area of research in the principal investigator’s laboratory. Furthermore, the combination of knowledge in molecular and computational biology will promote the training of young researchers in multidisciplinary approaches to complex biological challenges, contributing to Goal 4 of the 2030 Agenda: “Quality Education.” The project also adopts “Open Science” principles, promoting accessibility and collaboration in research.