A thymus-independent artificial organoid system supports complete thymopoiesis from Rhesus macaque-derived hematopoietic stem and progenitor cells

Thymic output has been extensively studied. While advanced ex vivo T cell generative approaches exist for mouse and human models, such advancements for nonhuman primate models are lacking. We report the establishment of a rhesus macaque-specific artificial thymic organoid (Rh-ATO) system, enabling the robust ex vivo generation of T cells from CD34⁺ hematopoietic stem and progenitor cells (HSPCs). A continuum of distinct thymopoietic stages was recorded - robust T cell specification of HSPCs resembling thymus seeding progenitors, emergence of CD4+CD3- immature single positive, CD4+CD8+ double positive thymocytes, and finally, generation of CD4⁺ and CD8⁺ single-positive T cell subsets expressing CD38, consistent with recent thymic emigrant phenotype. These events closely mirrored Bonafide thymopoietic stages observed in the thymus. T cells expressed TCRs and exhibited polyfunctional cytokine expression. Thus, we report the first demonstration of an off-the-shelf NHP-specific 3D system recapitulating thymopoiesis, providing a translational platform for modeling T cell development, therapeutic strategies, and immunopathogenesis.