Society: AGA

This session will provide the latest developmental research in human-derived organoids.

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.

Figure 1: Impaired Mitochondrial metabolism in Active UC (UC) colonoids compared to non-IBD Control colonoids (C). (a) A sample well ready for Seahorse MitoStress Test after 3 d differentiation. Scale bar= 200 μm. (b) Bioenergetic profile of active UC vs C colonoids. (c) Increased basal respiration and (d) Elevated proton leak in active UC vs C colonoids.

<b>Figure 2:</b> Elevated uncoupling protein 2 (UCP2) expression in colonoids from active UC vs non-IBD controls. (a) Rectal bulk RNA-seq data from the PROTECT Pediatric UC inception cohort study showed a significant upregulation of Ucp2 gene in UC rectal tissues vs non-IBD controls. n=20 for C, 206 for UC. P < 0.0001 from the Mann-Whitney test. (b) Ucp2 mRNA is more abundant in UC colonoids compared to control. (c) Representative immunofluorescent images of OCT-embedded colonoids stained with DAPI (nuclei), Phalloidin (F-actin), mitochondrial marker antibody (Cox4), and Ucp2 antibody. Arrows show co-expression of Cox4+Ucp2+ (yellow marks) in the merged image. Scale bar = 50 μm. UCP2 expression was quantified in 4 high-power fields per sample and expressed per nucleated cells. n=4 patients/grp. The line within the boxplot represents the median, + represents the mean, and the symbol above and below the whiskers represents the outliers that are either greater than the 95th or less than the 5th percentile.

Figure 2: Elevated uncoupling protein 2 (UCP2) expression in colonoids from active UC vs non-IBD controls. (a) Rectal bulk RNA-seq data from the PROTECT Pediatric UC inception cohort study showed a significant upregulation of Ucp2 gene in UC rectal tissues vs non-IBD controls. n=20 for C, 206 for UC. P < 0.0001 from the Mann-Whitney test. (b) Ucp2 mRNA is more abundant in UC colonoids compared to control. (c) Representative immunofluorescent images of OCT-embedded colonoids stained with DAPI (nuclei), Phalloidin (F-actin), mitochondrial marker antibody (Cox4), and Ucp2 antibody. Arrows show co-expression of Cox4+Ucp2+ (yellow marks) in the merged image. Scale bar = 50 μm. UCP2 expression was quantified in 4 high-power fields per sample and expressed per nucleated cells. n=4 patients/grp. The line within the boxplot represents the median, + represents the mean, and the symbol above and below the whiskers represents the outliers that are either greater than the 95th or less than the 5th percentile.

Maria Moreno-Fernandez

Appearances

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

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