Abstract: Reprogramming somatic cells in non-model species such as the rock hyrax (Procavia capensis) presents unique challenges due to divergent epigenetic landscapes and regulatory pathways. Here, we introduce a multi-stage reprogramming strategy that overcomes these barriers by integrating sequential genetic and chemical interventions. Our protocol combines transfection of pluripotency factors (OCT4, SOX2, KLF4, cMYC, NANOG, LIN28A, SV40, and GLIS1) with a tailored chemical cocktail targeting epigenetic modifiers and signaling pathways. Chromosomal stability was validated by karyotyping, and a portion of naïve-state markers were significantly elevated compared to non-transfected controls. The inclusion of stage-specific factors (e.g., GLIS1 priming, SV40 enhancement) and a defined small-molecule regimen addressing species-specific epigenetic resistance provides a novel framework for overcoming reprogramming bottlenecks. By enabling iPSC generation in the rock hyrax, a critical model for elephantid biology, this work advances comparative studies of genomic traits unique to Afrotherians and offers a potential platform for genetic engineering and conservation-based cellular resource development. While scalability and long-term functional assays require further exploration, this strategy establishes a foundation for reprogramming recalcitrant species, bridging gaps in evolutionary genomics and endangered species preservation.
