Journal
CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY
Volume 6, Issue 4, Pages 477-494Publisher
ELSEVIER INC
DOI: 10.1016/j.jcmgh.2018.08.001
Keywords
Microvillus Atrophy; Disease Modeling; Brush Border Formation; Apical Vesicular Transport
Categories
Funding
- French National Institute of Health and Medical Research (INSERM)
- Agence Nationale de la Recherche Hemophagocytic lymphohistiocytosis-Cytotox [ANR-12-BSV1-0020-01]
- LOEWE Center for Cell and Gene Therapy Frankfurt, Hessisches Ministerium fur Wissenschaft und Kunst
- Centre national de la recherche scientifique
- Ligue Regionale Contre le Cancer [22/29/35/72]
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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 Munc18-2IStxbp2-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 Munc18-2IStxbp2 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.
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