Patient-derived enteroids provide a platform for the development of therapeutic approaches in microvillus inclusion disease
Meri Kalashyan, … , Izumi Kaji, Jay R. Thiagarajah
Meri Kalashyan, … , Izumi Kaji, Jay R. Thiagarajah
Published August 29, 2023
Citation Information: J Clin Invest. 2023;133(20):e169234. https://doi.org/10.1172/JCI169234.
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Research Article Gastroenterology

Patient-derived enteroids provide a platform for the development of therapeutic approaches in microvillus inclusion disease

Abstract

Microvillus inclusion disease (MVID), caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid/base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient–derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel–blocking antidiarrheal drug crofelemer dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. γ-Secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum/glucocorticoid-regulated kinase 2 (SGK2) and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.

Authors

Meri Kalashyan, Krishnan Raghunathan, Haley Oller, Marie-Theres Bayer, Lissette Jimenez, Joseph T. Roland, Elena Kolobova, Susan J. Hagen, Jeffrey D. Goldsmith, Mitchell D. Shub, James R. Goldenring, Izumi Kaji, Jay R. Thiagarajah

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Figure 1

MVID enteroids recapitulate native epithelial disease changes.

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MVID enteroids recapitulate native epithelial disease changes. (A) Repre...
(A) Representative confocal images of villin (green), cytokeratin 20 (CK20, red), and nuclei (DAPI) of healthy and MVID (MYO5BKO) enteroids in cross section (left; scale bar: 10 μm) or en face (right; scale bar: 20 μm). Representative of 3 separate samples. (B) Representative electron micrographs of healthy and MVID enteroids (MYO5BKO). Representative of 2 separate samples. Scale bars: left, 2 μm; top right, 0.5 μm; bottom right, 1 μm. (C) Bright-field images of enteroid cultures grown in expansion versus differentiation medium. Inset images highlight changes in cultures with increased spheroid (stem-like) morphology in MVID differentiated cultures versus healthy. Representative of 6 separate experiments. (D) Enteroid formation assay showing new enteroid formation (at 4 days after plating) following enzymatic dissociation and replating. Data are shown as means ± SEM; n = 3 experiments; *P < 0.05, **P < 0.01; 2-way ANOVA with Tukey’s post hoc testing. (E) Relative gene expression (normalized to healthy expansion) for neurogenin 3 (NGN3), mucin 2 (MUC2), and alkaline phosphatase (ALPI) in healthy and MVID enteroids following switching to enteroid differentiation medium. Data are shown as means ± SD; n = 4 experiments; 2-way ANOVA with Tukey’s post hoc testing.