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 6

γ-Secretase inhibition rescues MVID patient enteroid differentiation.

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γ-Secretase inhibition rescues MVID patient enteroid differentiation. (A...
(A) Electron micrographs of healthy and MVID enteroids with and without DAPT treatment (10 μM). Scale bars: top panels, 0.5 μm; middle and bottom panels, 1 μm. (B) Analysis of electron microscopic images of microvillus length. (C) Subapical actin bundle length. (D) Distance of apical organelle free zone. Inset images above show example measured parameter. Graphs show measurements from at least 10 electron microscopic images. (E) Super-resolution confocal images (STED), en face (top) and cross section (bottom), of NHE3 localization and abundance in healthy and MVID patient enteroids following treatment with DAPT (10 μM). Scale bars: 5 μm. (F) Representative curves showing change in intracellular pH in healthy cells and MVID cells with and without DAPT. (G) Summary graph showing NHE3-dependent pH changes in healthy and MVID patient cells with and without DAPT. Data are shown as means ± SEM; n = 4–6 experiments; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; 2-way ANOVA with Tukey’s post hoc testing.