Genetic and microenvironmental determinants of epithelial quiescence, junctional stabilisation, and cytokine-burst duration at 2- and 5-day stages of human organoid maturation: a longitudinal clonal study

Background Timely entry into epithelial quiescence and controlled paracrine signalling are critical for early morphogenesis and subsequent regenerative competence. Nevertheless, the relative contributions of inherited variation and culture context to early epithelial quiescence, junctional stabilisation, and inflammatory-burst kinetics remain poorly resolved, as does the extent to which these influences remodel over the first days of organoid maturation. Methods We examined genetic and microenvironmental influences on (i) duration of epithelial quiescence, (ii) capacity to achieve junctional stabilisation, and (iii) length of the initial cytokine burst at 2 and 5 days post-seeding in 998 paired organoid cultures generated from mono- and dizygotic-equivalent induced pluripotent stem-cell clones. Classical twin modelling partitioned variance into additive genetic (A), shared microenvironmental (C), and culture-specific (E) factors. Polygenic scores were computed for epithelial integrity traits as well as a panel of congenital malformation risks. Results Additive genetic factors accounted for a substantial proportion of variance in cytokine-burst duration at both 2 and 5 days (A = 0.29–0.70) and in junctional stabilisation at 5 days (A = 0.51–0.67). Shared microenvironment dominated variability in re-entry from quiescence (frequency of cell-cycle reactivation) at both stages (C = 0.61–0.90) and in junctional stabilisation at 2 days (C = 0.36–0.65). Longitudinal modelling revealed modest shared genetic influence on daytime quiescence maintenance across stages (24%), whereas shared genetic effects on evening and nocturnal stabilisation metrics were non-significant. Shared microenvironment exerted moderate stable influence on quiescence re-entry events (56%), while cytokine-burst duration in evening and nocturnal windows displayed modest but significant shared genetic influence (17%–33%). Culture-specific effects were largely stage-specific. A polygenic score indexing risk for craniofacial dysmorphogenesis associated with prolonged evening cytokine burst at 2 days (β = 0.16, p = .002). Conclusions Etiological architecture shifted markedly between 2 and 5 days, underscoring a highly plastic window in early epithelial patterning and clone–microenvironment interplay. These findings highlight candidate genomic pathways and culture parameters that could be modulated to optimise regenerative outcomes in early human organoid systems.