Human 3D epithelioids enable continuous long-term clonal evolution studies across multiple epithelial tissues
Abstract
Modeling human epithelia in vitro remains challenging because current systems do not fully preserve the combination of architecture, heterogeneity, clonal composition, and long-term dynamics shown in vivo. While 3D approaches such as organoids and organotypic cultures capture important aspects of lineage differentiation and niche signaling, they often lose stable organization over time, limiting studies of long-lasting processes such as clonal evolution and cell competition. Here, we present human epithelioids as continuous long-term, 3D epithelial cultures efficiently derived from eight adult human epithelia, including trachea, skin, buccal mucosa, esophagus, blader, urethra, submandibular gland and endometrium. Using immunostaining, electron microscopy, single-cell RNA sequencing, functional assays and somatic mutation analyses, we deeply characterized human epithelioids and confirmed that they recapitulate native architecture and cell diversity, sustain regenerative capacity, and preserve donor-specific mutational landscapes, establishing a robust and versatile platform for longitudinal interrogation of clonal evolution, tissue dynamics and responses to clinically relevant perturbations, including radiotherapy and chemotherapy, over extended timescales.
Full-text reasoning
From the deep-tier full-text analysis of this preprint.
Reasoning review — 7 key claims, 6 well-supported, 1 with gaps
- supported Human epithelioids recapitulate the structural organization, cellular composition, and marker expression profiles of their corresponding tissues of origin across different epithelial subtypes.
- supported Human epithelioids exhibit tissue-specific ultrastructural features and epithelial integrity.
- unsupported The new protocol (ALI conditions, retained epithelial explants) achieves more robust long-term maintenance and improved differentiation of human epithelioids. gap: No comparative data is presented to show that the new protocol improves robustness, long-term maintenance, or differentiation compared to the previous method.
- supported Tracheal epithelioids display pseudostratified architecture with specific basal, ciliated, and secretory cell types and functions comparable to native tissue.
- supported Stratified epithelioids (skin, buccal, esophageal) reproduce characteristic squamous organization and differentiation markers.
- supported Urothelium epithelioids (bladder, urethra) recapitulate transitional stratified urothelium with specific cell layers and markers.
- supported Submandibular gland epithelioids contain ductal and acinar-like cells but lack the typical glandular tubular structure found in vivo.
Claims and gaps are read from the full text by a language model, shown for transparency; they do not affect ranking or selection.
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- biorxiv v1 2026-06-08 source ↗
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bioRxivpreprint @biorxivpreprint.bsky.social · 8893 followers neutral
Human 3D epithelioids enable continuous long-term clonal evolution studies across multiple epithelial tissues https://www.biorxiv.org/content/10.64898/2026.06.07.730560v1
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bioRxiv Cancer Bio @biorxiv-cancer.bsky.social · 3937 followers neutral
Human 3D epithelioids enable continuous long-term clonal evolution studies across multiple epithelial tissues https://www.biorxiv.org/content/10.64898/2026.06.07.730560v1