Abstract

Understanding how pluripotency regulatory networks evolve across mammals remains a central question in developmental and stem cell biology. While rodent models have defined the canonical core circuitry of pluripotency, the extent to which these regulatory hierarchies are conserved in large mammals is unclear. Here, we provide evidence that SOX15 contributes to the species-specific regulatory network as a modulator that sustains pluripotency and preserves functional differentiation capacity in porcine embryo-derived stem cells. Comparative sequence analysis revealed strong conservation of the SOX15 HMG domain across mammals, yet promoter divergence suggested lineage-specific regulatory evolution. Transcriptomic profiling demonstrated that, unlike in mice, SOX15 is robustly upregulated from the 2-cell stage and remains highly expressed in the porcine epiblast, coinciding with key windows of pluripotency establishment. In porcine embryo-derived stem cells, stable SOX15 knockdown resulted in reduced colony integrity, diminished alkaline phosphatase activity, impaired proliferation, and downregulation of core pluripotency genes, including OCT4 and NANOG. Furthermore, loss of SOX15 disrupts embryoid body formation and abolishes teratoma-forming capacity in vivo. This deficit likely reflects impaired pluripotency, but may also be attributed to compromised cell survival or proliferative fitness following transplantation. Notably, comparable perturbations in mouse models do not produce equivalent phenotypes, underscoring a lineage-dependent functional divergence. Together, our findings suggest that SOX15 contributes to the support of the porcine pluripotency network, and hint at evolutionary plasticity in the hierarchical architecture of mammalian pluripotency. These findings move beyond the rodent-centric paradigm and offer a refined perspective on the evolutionary plasticity of pluripotency networks, highlighting SOX15 as a pivotal lineage-specialized node governing naive pluripotency in large mammals. Notably, these inferences are based on functional perturbation using a single validated miRNAi construct; definitive confirmation of SOX15-specific causality will require future orthogonal validation via independent knockdown sequences, CRISPR interference, or RNAi-resistant rescue experiments.

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