Introduction: Interspecies chimeras using pluripotent stem cells (PSCs) offer a promising strategy to generate humanized tissues in organ-deficient pigs, potentially addressing the global shortage of donor organs. However, human PSCs have so far shown limited integration into porcine embryonic development. Expanded potential stem cells (EPSCs) resemble eight-cell and morula-stage blastomeres, exhibit robust proliferation, allow precise genome editing, and possess enhanced differentiation potential. These features make them strong candidates for improving chimera formation and modeling early development. To investigate interspecies cell competition, we established (1) a porcine-human EPSC co-culture system and (2) a porcine EPSC-derived “heart niche” model to study in vitro competitive interactions during early cardiac co-differentiation.
Methods: For the in vitro interspecies co-culture assay, GFP-expressing human primed iPSCs were stepwise converted into EPSCs and assessed for the upregulation of pluripotency and core histone genes. Equal numbers of single-cell dissociated porcine tdTomato⁺ EPSCs and human GFP⁺ EPSCs were co-cultured to evaluate proliferation, cell-cell communication, and apoptosis until confluency. The mixed cultures were then differentiated into cardiomyocytes using a protocol optimized for both human and porcine EPSCs. Additionally, NKX2.5 knockout porcine EPSCs were generated using the CRISPR-Cas9 system to study cell competition with human EPSCs during cardiac co-differentiation.
Results: We developed an interspecies EPSC co-culture system to study in vitro cell competition between porcine and human EPSCs. Under optimized expanded potential conditions, both cell types proliferated efficiently and maintained stable dome-shaped colonies. Time-lapse microscopy and immunostaining for PARP and Caspase revealed no evidence of interspecies competition. During co-culture, porcine and human cells formed segregated, adjacent colonies connected via E-cadherin at their borders. Upon directed cardiac differentiation, the mixed cultures produced integrated, beating cardiomyocytes. Notably, NKX2.5 knockout porcine EPSCs displayed impaired cardiac differentiation, leading to fewer cardiomyocytes and dominance of human EPSC-derived cardiomyocytes in interspecies co-differentiation. 
Conclusion: Due to their enhanced pluripotency and differentiation potential, porcine EPSCs provide a robust platform for in vitro modeling of interspecies chimerism and for the pre-evaluation of gene candidates aimed at generating niche-enabled pigs for the production of humanized organs.