Background & aims: Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID.
Methods: Intestinal organoids from Munc18-2/Stxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions.
Results: Loss of Munc18-2/Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin-positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane.
Conclusions: We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border.
Keywords: 3D, 3-dimensional; Apical Vesicular Transport; Brush Border Formation; DAPI, 4′,6-diamidino-2-phenylindole; Disease Modeling; EGFP, enhanced green fluorescent protein; FHL5, familial hemophagocytic lymphohistiocytosis type 5; IWP-2, inhibitor of WNT production-2; KO, knock-out; MVID, microvillus inclusion disease; MVIs, microvillus inclusions; Microvillus Atrophy; PBS, phosphate-buffered saline; STXBP2, syntaxin binding protein 2; Stx3, syntaxin 3; TEM, transmission electron microscopy; VPA, valproic acid; WT, wild-type.