A xeno-free human iPSC-derived prostate organoid platform for multilineage differentiation and genetic manipulation.

Current prostate organoid models rely on tissue-derived material or animal components and lack epithelial and stromal complexity. We defined a xeno-free system to generate human prostate organoids from induced pluripotent stem cells with consistent multilineage differentiation. Organoids formed as free-floating 3D aggregates self-organized into the epithelial and stromal domains with basal, luminal, neuroendocrine, fibroblast, and smooth muscle markers. In an alternative modular co-culture system, engineered epithelial progenitors aggregated with wild-type mesenchymal progenitors, enabling compartment-specific manipulation. Androgen receptor (AR)-overexpressing organoids showed increased epithelial AR and prostate-specific antigen (PSA) expression and proliferation. Single-cell transcriptomics, together with qPCR and immunostaining, confirmed prostate lineage specification and tissue organization. This xeno-free platform provides a reproducible, scalable, and genetically tractable model to study in vitro prostate lineage programs, epithelial and stromal interactions, and disease biology.