Unique Human Intestinal Tuft Cell Features Revealed by Pluripotent Stem Cell-Derived Mini-Guts.

BACKGROUND AND AIMS: Intestinal tuft cells are rare chemosensory epithelial cells critical for mucosal immunity. However, the molecular features of human intestinal tuft cells remain incompletely defined due to limited access to human tissue and known species-specific differences from murine models. We sought to establish a human tuft cell-enriched intestinal organoid system derived from pluripotent stem cells. METHODS: Human pluripotent stem cell-derived intestinal organoids containing epithelial, stromal, and neural components were subjected to Notch pathway inhibition and T helper 2 cytokine stimulation to promote tuft cell differentiation. Induced tuft cells were analyzed by immunofluorescence, quantitative polymerase chain reaction (qPCR), and single-cell RNA sequencing, followed by integrated transcriptomic comparison with primary human small intestinal tuft cells. RESULTS: Tuft cell-enriched organoids were reproducibly generated from 3 independent human pluripotent stem cell lines. Integrated single-cell transcriptomic analyses demonstrated strong concordance between in vitro-derived and primary human tuft cells. The organoid-derived tuft cells exhibited distinct molecular features, including low and non-enriched interleukin-25 expression and predominant DCLK2 expression, distinguishing them from murine tuft cells. Under defined experimental conditions, murine-reported regulatory mechanisms, including bone morphogenetic protein-dependent negative feedback and goblet cell-mediated lateral inhibition, were not recapitulated. CONCLUSION: We establish a human pluripotent stem cell-derived intestinal organoid model that enables enrichment and molecular characterization of human tuft cells. This platform provides a human-specific system for elucidating molecular programs governing tuft cell differentiation and epithelial-immune signaling.