Naïve culture environments differentially shape human endogenous retrovirus-K expression and transcriptional remodeling following transient naïve treatment of human induced pluripotent stem cells

Human endogenous retroviruses (HERVs) are developmentally regulated transposable elements whose expression changes with pluripotent-cell state. Among them, HERV-K and its associated LTR5_Hs elements provide informative readouts of transcriptional resetting during hiPSC reprogramming. Transient naïve treatment has been proposed to improve the molecular fidelity of human induced pluripotent stem cells (hiPSCs), but naïve pluripotency can be induced using culture systems that engage different signaling and epigenetic regulators. Whether these environments exert equivalent effects on HERV activity, and the host transcriptome remains unclear. Here, we subjected established hiPSCs to transient naïve treatment using 4CL, expanded-potential stem cell (EPS), PXGL, and t2iLGoY environments. In the initial t2iLGoY-based PNP experiment, the normalized expression of HERV-K gag, env, pol, and LTR5_Hs was lower than that in time-matched hiPSCs. Across the four primed-naïve-primed (PNP) conditions, however, these HERV-K-associated targets showed condition-associated expression patterns. Comparison across the four environments revealed variation in the magnitude and pattern of HERV-K responses, including discordant regulation of HERV-K coding transcripts and LTR5_Hs. RNA sequencing further showed that the resulting cells occupied distinct host transcriptional states rather than converging on a common human embryonic stem cell-like endpoint. These states were accompanied by condition-specific pluripotency-associated expression patterns and enrichment of different developmental gene programs. Our findings indicate that naïve medium composition is associated with distinct transcriptional outcomes following PNP treatment.