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

Abstract 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. Floating organoids self-organize into epithelial and stromal domains with basal, luminal, neuroendocrine, fibroblast, and smooth muscle markers. In an alternative modular co-culture system, engineered epithelial progenitors are aggregated with wild-type mesenchymal progenitors, enabling compartment-specific manipulation. Androgen receptor–overexpressing organoids showed increased epithelial AR and PSA expression and proliferation. Single-cell transcriptomics, together with qPCR and immunostaining, confirmed prostate lineage specification and tissue organization. This new xeno-free platform provides a reproducible, scalable, and genetically tractable model to study in-vitro prostate lineage programs, epithelial-stromal interactions, and disease biology. Graphic Abstract This study describes the generation of prostate organoids from human iPSCs. iPSCs, including those reprogrammed from patients carrying germline mutations, can be differentiated into prostate organoids either through monoculture or by co-culturing endodermal cells with mesenchymal progenitors. Genetic manipulation can be introduced before endoderm specification to model cancer drivers. The resulting multi-lineage organoids exhibit distinct epithelial (AR⁺, NKX3.1⁺, PSA⁺, CK8/18⁺) and stromal (VIM⁺, α-SMA⁺) compartments, providing a versatile platform for developmental studies, disease modelling, drug screening, and biomarker discovery.