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 5

Crofelemer inhibits chloride and fluid secretion in MVID patient enteroids.

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Crofelemer inhibits chloride and fluid secretion in MVID patient enteroi...
(A) Representative curve showing dose-dependent inhibition of forskolin- (10 μM) and carbachol-stimulated (50 μM) Isc by crofelemer. (B) Dose-response curve for crofelemer-induced inhibition of forskolin- and carbachol-stimulated Isc in MVID patient enteroids (MYO5B KO). Data are shown as means ± SEM; n = 6 monolayers. (C) Maximal percentage inhibition of agonist-stimulated current by crofelemer. Data are shown as means ± SEM; n = 6 monolayers. (D) Example bright-field images before and after forskolin (2 μM) ± crofelemer (200 μM) in MVID patient enteroids. Right panels show higher magnification of enteroid swelling. Representative of 4 separate experiments. Scale bars: left panels, 1 mm; right panels, 250 μm. (E) Violin plot showing increase in enteroid size (diameter ratio) in healthy and MVID enteroids at 1 hour ± forskolin and ± crofelemer (200 μM). Dotted lines, median ± interquartile range; >300 enteroids from 4 experiments. (F) Diameter ratio in MVID enteroids at 2 and 4 hours after stimulation ± crofelemer (200 μM). Dotted lines, median and interquartile range; n = 3 experiments; *P < 0.05, **P < 0.01, ***P < 0.001; 2-way ANOVA with Tukey’s post hoc testing.