Investigating genetic and epigenetic regulators of human naive pluripotent stem cells

Pluripotent stem cells can exist in two primary cellular states referred to as naive or primed. Naive pluripotent stem cells possess enhanced developmental potency compared to conventional primed states. However, generating and maintaining human naive pluripotent stem cells remains challenging. Developing tools to isolate naive cells and elucidate associated genetic and epigenetic regulators is therefore essential. We aimed to create a reporter for isolating naive cells from heterogeneous cultures, optimize naive induction protocols, and uncover regulators of naive status acquisition. Based on expression data, we selected KLF17 as a gene specifically expressed in naive pluripotent stem cells. Therefore, we engineered a KLF17-T2A-EGFP fluorescent reporter in human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9-based genome editing. We tested this GFP-based reporter system during naive conversion steps. Quantitative PCR analysis of EGFP expression showed naive-specific activation, indicating successful construction of the reporter. Optimizing the conversion of naive stem cells is an ongoing challenge. We tested the effects of overexpressing KLF17 and NANOG/LIN28A on naive induction. qPCR results demonstrated these transcription factors increased activation of naive markers compared to controls, suggesting they may accelerate resetting. We also aimed to assess the effects of KLF17 and NANOG overexpression on naive and primed reprogramming efficiency from somatic cells. The results showed that overexpression of KLF17 did not affect reprogramming efficiency. However, NANOG overexpression increased the efficiency of both primed and naive reprogramming. In addition, we investigated the impacts of knocking out transcription factors KLF5, KLF17, and LIN28A on naive and primed reprogramming. KLF5 knockout did not affect either reprogramming system. But KLF17 knockout specifically decreased naive reprogramming efficiency while not affecting primed reprogramming. In contrast, LIN28A knockout reduced the efficiency of both primed and naive reprogramming. Lastly, we tested small molecule inhibitors of epigenetic regulators on primed and naive reprogramming. The inhibitors iCBP112, iBRD9, RN1, A83-01, and EPZ5676 all significantly increased reprogramming efficiency under both primed and naive conditions. Taken together, these results demonstrated that both transcription factors and epigenetic regulators affect the acquisition of primed vs. naive identity from somatic cells.