Characterization of chemical and genetic regulators of human naive pluripotent stem cells

Pluripotent stem cells exist in two main developmental states, which are naive and primed. Naive pluripotent stem cells hold particular promise for modelling early embryonic development due to their broader developmental potential. However, the derivation and stable maintenance of human naive pluripotent stem cells remain technically challenging. This thesis aims to uncover the epigenetic mechanisms that restrict the transition from the primed to the naive state and to develop strategies that improve conversion efficiency. In particular, the effects of several chromatin modifying factors (DOT1L, P300/CBP, MENIN, and BRD9) on naive conversion were evaluated using small-molecule inhibitors. Two independent reporter systems (one based on differential enhancer usage of OCT4 and the other based on KLF17 expression) were employed to monitor naive conversion. Inhibitor treatments were conducted under a recently developed culture condition (HENSM). Conversion efficiency was assessed quantitatively using flow cytometry for fluorescent marker expression and RT-qPCR for naive-specific genes. The findings demonstrate that DOT1L inhibition using EPZ5676 and MENIN inhibition using VTP50469 significantly relieve epigenetic constraints on naive conversion. On the other hand, inhibitors targeting P300/CBP and BRD9 exhibited more limited effects. RT-qPCR results confirmed the upregulation of naive markers such as DPPA3, TFCP2L1, and KLF17, and the downregulation of primed or lineage-related genes. Collectively, these results indicate that DOT1L and MENIN function as key epigenetic barriers to naive pluripotency in human stem cells. In addition, a novel KLF17-GFP reporter line, generated in this laboratory, was functionally characterized in this thesis to validate its utility as a naive-specific marker for monitoring state transitions. In the third part of the thesis, to facilitate gene knockouts in both primed and naive iPSCs, I generated a human iPSC line carrying an inducible Cas9 gene at the AAVS1 safe harbour locus via genome editing. Overall, this study shows that inhibiting specific epigenetic regulators like DOT1L and MENIN can greatly improve the efficiency of generating human naive pluripotent stem cells. It also introduces a novel reporter system and a genome-edited iPSC line that can be suitable for dissecting the regulation of naive pluripotency and for gene knockout studies in both naive and primed conditions.