PATIENT-DERIVED ORGANOIDS REVEAL IMPAIRED MITOCHONDRIAL BIOENERGETIC EFFICIENCY IN THE COLON EPITHELIA OF PEDIATRIC PATIENTS WITH ULCERATIVE COLITIS

Background: Colon epithelial mitochondrial (MT) dysfunction is implicated in the pathogenesis of pediatric UC. Patient-derived organoids recapitulate several complexities of the parent tissue, but it is unknown if they can model the mitochondrial functional impairments observed in the UC epithelium. The aim of this study is to identify functional metabolic derangements unique to the pediatric UC epithelium using patient-derived colon organoids (colonoids) as a model of human UC.

Methods: We generated colonoids from rectal biopsies obtained from pediatric patients with endoscopically active and inactive UC, and non-IBD controls [n=4/grp, median age 14.5 yrs]. Cryopreserved colonoids were thawed, passaged, and differentiated for 3 days. We analyzed the bioenergetics of colonoids (in quadruplicate) in real-time through Seahorse Analyzer MT stress testing. We used gene expression analysis, imaging, spectrometric, and flow cytometric methods to assess MT membrane potential (MMP), MT-reactive oxygen species (ROS) production, MT-mass, and MT-uncoupling proteins.

Results: At baseline, colonoids from active UC patients consumed more oxygen than those from non-IBD controls (P=.021), but produced a similar amount of ATP. Accordingly, we found an 85% increase in proton leak and lower MMP (assessed by JC-1 uptake) in active UC compared to non-IBD colonoids (P=.015) (Figure 1). Moreover, MT-ROS production was higher in active UC compared to non-IBD colonoids. In line with the observed higher proton leak in active UC, we found a significantly increased gene and protein expression of the MT respiratory chain uncoupling protein Ucp2 in active UC compared to non-IBD colonoids (P=.030 and .024, respectively, Figure 2). We validated the observed elevated Ucp2 in active UC through the analysis of rectal bulk RNA-seq data from the PROTECT Pediatric UC inception cohort (GSE109142). In the stem cell state before differentiation, MT-mass (normalized to 7-AAD^- colonoid single cells) was similar among all colonoid groups. After differentiation, active UC colonoids exhibited higher mitochondrial mass than non-IBD controls (P=.019). Overall, colonoids from inactive UC patients showed similar metabolic characteristics to non-IBD controls.

Conclusion: Colonoids derived from active pediatric UC patients demonstrate inefficient MT bioenergetics marked by higher proton leak and the requirement of more oxygen to generate ATP compared to those from non-IBD patients. Such MT inefficiency may be caused by observed higher expression of uncoupling protein or increased ROS generation in UC colonoids. These data demonstrate that UC epithelial MT functional impairment can be modeled in patient-derived colonoids and support advancing colonoids as a preclinical human model for testing therapies against such metabolic dysfunction.