| Popis: |
Human pluripotent stem cells (hPSCs) show great potential for regenerative medicine applications, but several limitations must be addressed before their widespread clinical use. These limitations include epigenetic instability, which can lead to spontaneous differentiation or genomic instability, and heterogeneity among hPSC lines, resulting in cells with different and unexpected properties. In addition, there are safety concerns regarding the potential for teratoma formation, immune rejection, and prolonged culture of hPSCs–which may result in X chromosome-linked erosion, referring to the gradual loss of the silencing of one of the two X chromosomes in female cells. This erosion can cause increased expression of X-linked genes, affecting developmental and physiological consequences that can limit their differentiation potential and, consequently, their therapeutic efficacy. To overcome these limitations, it is necessary to improve our understanding of the genetic and epigenetic mechanisms that regulate hPSCs and develop culture conditions that more closely resemble the in vivo environment. Recent advances in human naïve culture conditions offer potential solutions for these challenges. Naïve human pluripotent stem cells maintained in culture resemble pre-implantation epiblast cells and represent an earlier developmental state than conventionally cultured hPSCs, which are in a primed state of pluripotency and resemble post-implantation epiblast cells. Compared to naïve cells, conventional hPSCs have a more restricted developmental potential and are closer to differentiation. We recently developed new culture conditions that allow naïve hPSCs to transition to the primed state of pluripotency within a time frame that mimics early development. We refer to this transition as "capacitation". However, it is uncertain whether capacitated naïve hPSCs can accurately replicate the gene expression and epigenetic patterns observed in the primed state after acquiring multi-lineage differentiation ability. Furthermore, it is unknown whether resetting conventionally cultured primed hPSCs to the naïve state and then allowing them to capacitate in the primed state could potentially address epigenetic anomalies such as X-chromosome inactivation erosion. Aiming to clarify these questions, we employed sequencing assays to characterize and model gene expression, DNA methylation, histone modifications, and chromatin accessibility landscapes of naïve, capacitated, and long-term cultured hPSCs. This dissertation presents and interprets the results of this multi-omics analysis, intended to enhance our understanding of the role of epigenetics in pluripotency, identify new targets for future research, and determine the advantages of naïve hPSCs capacitation. The results show that resetting conventionally cultured primed hPSCs to the naïve state followed by repriming restores the epigenetic characteristics of X chromosome inactivation from an eroded landscape. Additionally, they emphasise the importance of CpG islands, enhancers, and retrotransposons as hotspots of epigenetic dynamics between pluripotency states. Finally, they highlight the putative role of H3K27ac modifications in promoters of genes with naïve-specific expression. This study underscores the potential of improved hPSC culture methods for developing novel regenerative therapies and provides insights into the molecular mechanisms underlying epigenetic changes during capacitation. These findings have implications for generating clinically relevant cell types and contribute to the ongoing progress in regenerative medicine, laying the foundation for future investigations in optimizing human pluripotent stem cell culture methods. |
